Abstracts for the 60Th Annual Meeting of the Society for Investigative Dermatology Sheraton Chicago Hotel, Chicago, Illinois May 5–9, 1999

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ABSTRACTS Abstracts for the 60th Annual Meeting of the Society for Investigative Dermatology Sheraton Chicago Hotel, Chicago, Illinois May 5–9, 1999

Plenary and Concurrent Sessions Abstract Numbers Plenary and Concurrent Sessions Abstract Numbers

Plenary Session I 0001–0016 Posters Concurrent Minisymposia Aging 256–262, 542–548 Use of Dendritic Cells as Immunotherapeutic Agents 001–008 Bullous Disease 263–277, 549–563 Hair Follicle Morphogenesis/Carcinogenesis 009–016 Carcinogenesis 278–288, 564–574 Apoptosis 017–024 Cell Biology/Biochemistry 289–300, 575–585 Adhesion: Components and Mechanisms 025–032 (General) General Plenary Session 1 033–049 Cell Biology/Biochemistry 301–336, 586–621 Concurrent Oral Sessions (Keratinocyte) Bullous Disease 050–062 Cell Biology/Biochemistry 337–348, 622–632 Immunology/Inflammation–Cytokines,SolubleFactors 063–074 (Langerhans Cell/Lymphocyte) Extracellular Matrix/Connective Tissue 075–087 Cell Biology/Biochemistry 349–359, 633–642 Epidemiology/Health Services Research 088–100 (Melanocyte) Cell Biology/Biochemistry–Melanocytes 101–113 Dermatopathology/Morphology 360–371, 643–654 Lipids/Barrier Function 114–126 Extracellular Matrix/Connective 372–388, 655–672 Cell Biology/Biochemistry 127–139 Tissue Gene Expression 389–406, 673–689 General Plenary Session 2 140–154 Genetic Disease/Gene Therapy 407–424, 690–708 Concurrent Oral Sessions Growth Factors/Signal 425–438, 709–722 Gene Expression 155–166 Transduction Genetic Disease/Gene Therapy 167–178 Immunology/Inflammation 439–459, 723–743 Growth Factors/Signal Transduction 179–190 (Cellular) Immunology/Inflammation–Cellular 191–202 Immunology/Inflammation 460–481, 744–766 Carcinogenesis 203–215 (Cytokines, Soluble Factors) Cell Biology/Biochemistry 2 216–227 Photobiology 228–239 Infectious Disease/Virology 482–490, 767–774 Concurrent Minisymposia Lipids/Barrier/Percutaneous 491–467, 775–780 Growth Factors/Signal Transduction 240–247 Absorption Novel Therapeutic Strategies/Clinical Trials 248–255 Pharmacology/Clinical Trials 498–518, 781–800 Photobiology 519–541, 801–823 Epidemiology/Health Services Research 824–839

Committee on Scientific Programs, 1998–1999 Jack B. Longley, Thomas A. Luger, Kathi C. Madison, Estela E. Medrano, John C. Ansel, Leonard M. Milstone, Alice P. Pentland, Dennis R. Roop, Robert L. Modlin, Pauline M. Schwartz, Glynis Scott, Georg Stingl, Raija Chairman, Tung-Tien Sun, David R. Bickers (ex officio), David A. Norris Tammi, Maria L. Turner, Mark C. Udey, Martin A. Weinstock (ex officio) Exhibitors at the Annual Meeting Ad Hoc Reviewers American Skin Association, Inc., Blackwell Science, Inc., Candela Corpora- Elliot J. Androphy, Cheryl A. Armstrong, Angela M. Christiano, Ponciano tion, Dow Pharmaceutical Sciences, Ferndale Laboratories, Inc., Galderma D. Cruz, Jr., Vincent A. Deleo, Andrzej Dlugosz, Richard L. Eckert, James Laboratories, Inc., Spex Industries, Shandon Lipshaw, Coalition of Patient T. Elder, Craig A. Elmets, Kenneth R. Feingold, Richard L. Gallo, Barbara Advocates for Skin Disease Research, National Alopecia Areata Foundation, A. Gilchrest, Kathleen J. Green, Anne R. Haake, Russell P. Hall III, National Foundation for Ectodermal Dysplasias, PXE International, Inc., Robin Hornung, Pamela J. Jensen, Paul A. Khavari, Robert M. Lavker, Sturge-Weber Foundation

Abstracts for the Annual Meeting of the International DermatoEpidemiology Association Sheraton Chicago Hotel, Chicago, Illinois May 6, 1999

Plenary and Concurrent Sessions Abstract Numbers Plenary and Concurrent Sessions Abstract Numbers

Oral Session I01–I08 Poster Sessions (See SID Posters ‘‘Epidemiology/Health Services Inﬂammatory and Infectious Diseases I01–I02 Research’’ in this volume of the JID.) Skin Cancer/Melanoma I03–I06 Inﬂammatory and Infectious Diseases 824–832 Health Care Research I07–I08 Skin Cancer/Melanoma 833–835 Health Care Research 836–839

001 002 Dendritic Cell Migration from Blood into Inflamed Skin Sites Prophylaxis and Therapy of Ongoing Immune Responses by IL-10-Treated Dendritic Cells In Vivo S. Grabbe,*† C. Robert,* R. C. Fuhlbrigge,* and T. S. Kupper* A. H. Enk, G. Mu¨ller, H. Jonuleit, K. Steinbrink, T. Tu¨ting, C. Szalma and J. Knop *Harvard Skin Disease Research Center, Harvard Medical School, Boston, Massachusetts; and Department of Dermatology, University of Mainz, Mainz, Germany †Department of Dermatology, University of Mu¨nster, Mu¨nster, Germany Treatment of immature dendritic cells (DC) with IL-10 is able to convert these antigen-presenting According to current paradigms, dendritic cells (DC) that reside in peripheral tissues internalize cells into tolerizing antigen-presenting cells in vitro. In order to assess whether IL-10-treated DC and present antigen both for priming of naı¨ve T cells as well as for elicitation of cell-mediated would also be able to induce tolerance in vivo we used a system of OVA-TCR-tg mice (DO11.10). immune responses. Since both mature DC and DC precursors are present in peripheral blood, we In these animals 40%–70% of all naı¨ve T cells express an ovalbumin-specific T cell receptor. To tested the hypothesis that circulating antigen-presenting cells may infiltrate inflamed skin and assess whether IL-10-treated DC would be able to prevent sensitization in these mice in vivo, thereby contribute to elicitation of antigen-specific cutaneous immune responses. For this purpose, murine bone-marrow derived DC were cultured fetal calf serum-free with GM-CSF/IL-4 and DC of various maturation stages were generated from murine bone marrow by culture in GM- treated with 30 ng rmIL-10 per ml starting at day 2 of culture. On day 5 cells were pulsed with CSF Ϯ IL–4 Ϯ LPS and labeled with either a radioactive (51Cr) or a fluorescent (calcein) marker. 50 µg oxazolone (OVA) per ml. OVA-pulsed IL-10-DC and Ag-pulsed control DC, as well as Labeled DC were then injected i.v. into mice that were either sensitized and challenged with the unpulsed DC were injected into the tail vein of DO11.10 mice. Eight days later, mice were hapten oxazolone or challenged with the irritant phorbol ester PMA. Six hours later, ears and subcutaneously immunized with 30 µg OVA-peptide in IFA. Again 10 d later, total lymph node lymphatic organs were excised and analyzed for the presence of labeled DC. A small but significant cells (LNC) were harvested and restimulated with the OVA-peptide or a control peptide in vitro. immigration of circulating DC into inflamed skin was observed. No specific label was found in While good proliferative responses were observed in animals pretreated with Ag-pulsed DC or regional lymph nodes 6 h after injection, or in the skin after injection of nonviable DC or control unpulsed DC or even in controls that were only immunized subcutaneously, LNC from mice cells. DC extravasated into inflamed skin about five times more efficiently than into uninflamed that had received IL-10-treated Ag-pulsed DC showed only minimal response to peptide challenge. skin. Immature DC, generated by incubation in GM-CSF ϩ IL-4 for 4 d, as well as the immature Thus pretreatment of OVA-tg mice with IL-10-DC prevents sensitization in vivo. To see, whether DC line XS52, homed most efficiently to sites of allergic contact dermatitis and less well to sites IL-10-DC can also downmodulate ongoing immune responses, DO11.10 mice were sensitized of irritant dermatitis. Preincubation of DC with TGFb1 further enhanced homing of immature by s.c. injection of OVA-peptide in IFA. Eight days later, T cells from sensitized tg-mice were DC to inflamed skin. Interestingly, mature DC lost their capacity to home to inflamed tissues. harvested and sorted for OVA-TCR T cells. Five ϫ106 of these T cells were injected i.v. into DC homing was observed as early as 2 h after oxazolone challenge and peaked at 6–8 h. normal BALB/C mice. Again 2 d later, these animals received either OVA-pulsed IL-10DC, Extravasation of DC into inflammatory skin sites was clearly detectable by confocal microscopy OVA-pulsed, or unpulsed control DC. Seven days later, total LNC from these animals were of anti-CD31-labeled cutaneous vessels and calcein-labeled DC. Thus, dendritic antigen-presenting harvested and restimulated with OVA peptide in vitro. LNC from animals from control mice cells extravasate from blood into inflamed skin and may thus play a role in elicitation or persistence showed a significant proliferation towards OVA peptide, while proliferation in T cells from mice of T cell-mediated skin disorders. pretreated with IL-10-DC was inhibited by 70%. These data indicate that IL-10-DC are able to downmodulate ongoing immune reactions in vivo. Further experiments are necessary to explain the mechanism of inhibition or tolerance in this system.

003 004 Anti-Tumor Immunity Induced by Immunization with RNA-Pulsed Epidermal Cells Dendritic Cells Function as Antigen-Presenting Cells in GM-CSF-Based Melanoma Vaccines R. D. Granstein and W. Ding A. Schneeberger, P. Lu¨hrs, R. Kutil, H. Schild,* P. Steinlein,† and G. Stingl Department of Dermatology, Weill Medical College of Cornell University, New York, New York DIAID, Department of Dermatology, University of Vienna Medical School, Vienna, Austria; RNA-pulsed dendritic cells can reportedly be used to immunize against antigens encoded for by †Research Institute of Molecular Pathology, Vienna, Austria; *Department of Immunology, the RNA utilized. As the epidermis contains dendritic antigen-presenting cells (Langerhans cells) Laboratory of Cell Biology, University of Tu¨bingen, Germany and is a convenient site for immunization strategies, we asked if epidermal cells (EC) enriched for We and others could recently demonstrate that the injection of GM-CSF-expressing cancer cells Langerhans cell content could be used to immunize mice against the S1509a spindle cell tumor into experimental animals leads to a protective, T cell-mediated antitumor immune response. The a a/d (H-2 ) after pulsing with S1509a-derived RNA. EC from CAF1 mice (H-2 ) were enriched for goal of the present study was the delineation of the mechanism(s) resulting in the activation of Langerhans cell content by treatment with anti-Thy 1.2 antibodies and complement (eEC). eEC the specific T lymphocytes. were cultured for 16 h in 50 U GM-CSF per ml and then pulsed with 10 µg total cellular RNA In a first set of experiments, we used a contact hypersensitivity system (CHS), a classical model per ml from S1509a cells or from NS cells (a BALB/C-derived fibroblast line). eEC were then for T cell-mediated immunty. GM-CSF-secreting M3 melanoma cells (M3-GM; H-2d) or bone 5 washed 3´ and 2 ϫ10 cells injected s.c. into each of five CAF1 mice. Control mice were marrow-derived dendritic cells (BMDC) were haptenized and then s.c. injected into either immunized with eEC not exposed to RNA or with cells pulsed with a soluble extract of S1509a syngeneic or allogeneic hosts. Five days later, the recipients’ CHS response was tested by cells as a source of tumor-associated antigens (TAA) (pos control). Priming was repeated weekly ϫ2 epicutaneous hapten administration. Haptenized BMDC (H-2d) elicited a CHS response in and 1 wk later, all mice were challenged s.c with 1.5 ϫ106 live S1509a cells. Tumor growth was syngeneic, but not in allogeneic recipients (H-2b, H-2k), compatible with the expected mode of scored over time. Mice immunized with eEC pulsed with S1509a RNA had significantly reduced direct antigen presentation. By contrast, haptenized M3-GM cells were able to induce an ear tumor growth compared to the groups immunized with eEC pulsed with NS cell RNA or eEC swelling response in syngeneic as well as in allogeneic mice, arguing for a critical role of host not pulsed with RNA or TAA [at day 14: 363 mm3 for eEC ϩ S1509a RNA, vs 2330 mm3 for antigen-presenting cells (APC). eEC ϩ NS RNA (p Ͻ 0.01), vs 2078 mm3 for eEC alone (p Ͻ 0.01), vs 384 mm3 for medium To identify these APC, we introduced the aˆgal gene into M3-GM cells. The s.c. inoculation of alone (NS)]. In preliminary experiments, priming with eEC depleted of Langerhans cells by these cells into syngeneic DBA/2 mice induced a bgal-specific, MHC class I-restricted T cell antibody and complement mediated lysis prior to pulsing with S1509a RNA or with eEC pulsed response. We then assessed lymph nodes draining M3-GM-bgal injection sites for the presence of with RNA degraded with RNase failed to induce significant antitumor immunity while bgal epitope-bearing cells using a specific T cell clone. Results obtained showed that lymph nodes immunization with eEC pulsed with RNase-treated TAA did induce significant immunity. In draining M3-GM-bgal-, but not those of M3-GM injection sites contain cells able to stimulate another preliminary experiment, immunity against the S1509a tumor appeared to be induced by the bgal-specific CTL clone to produce IFN-a. Time kinetic studies revealed that these cells three weekly s.c. injections of total cellular RNA from S1509a. Experiments to exclude the appear by day 3, peak by days 4–5 and are detectable until day 6. Employing positive selection possibility of contaminating protein antigens have not yet been performed in this latter protocol. techniques (MACS-and FACS-sorting), we found that the antigen-presenting activity resides eEC pulsed with S1509a RNA can immunize naı¨ve mice against the S1509a tumor. within the CD11c (N418)-positive lymph node cell population. Together, these data demonstrate for the first time that DC represent the critical APC in GM- CSF-based cancer vaccines.

005 006 Cathepsin S Activity is Essential for Rapid Invariant Chain Processing and MHC Class II Peptide Assessment of Bone-Marrow Derived Dendritic Cells for Protein and Contact Hypersensitivity Loading in Human Dendritic Cells E. Olasz, N. Yamada, J. Linton and S. I. Katz D. Maurer, E. Fiebiger, I. F. Feng, B. Reininger and G. Stingl Derm Branch, National Cancer Institutes, Bethesda, Maryland DIAID, Department of Dermatology, University of Vienna Medical School, Vienna, Austria Because of their potent antigen-presenting capacity, dendritic cells (DC) have been identified as The exceptional capacity of dendritic cells (DC) to present exogenous antigen is linked to their having tremendous potential for use in immunotherapy protocols. As these protocols require large remarkable antigen uptake and processing functions and their ability to efficiently display peptide- numbers of DC, we utilized a model that relies on cytokine-expanded (GM-CSF and IL-4) bone bound MHC class II ab complexes (class IIab-peptide) along with costimulatory signals for T cell marrow cells (BALB/C and B6) as a source of DC. The purpose of this study was to functionally activation. These cellular functions are subject to regulation by external stimuli, e.g. cytokines, characterize these DC (Class II, CD80, 86, 11b, 11c, 40 positive, and CD14 negative) in vitro (in which can switch the DC’s program from the default antigen capture to the presentation mode. protein-antigen and allo-specific assays) and in vivo (using protein antigen or hapten-pulsed DC) Data obtained so far suggest that cytokine-activated DC efficiently display class IIab-peptide due to determine their suitability for the generation of T helper cell responses. Coculture of protein- to the increased synthesis and enhanced stability of MHC class II moieties. Here we show that pulsed (with hen egg lysozyme-HEL-or pigeon cytochrome c-CYTOc) DC with T cells from cytokine-activated human DC not only stabilize class IIab-peptide but also channel newly HEL/CFA or CYTOc/CFA-sensitized mice induced antigen-specific T cell proliferation, but was synthesized class II-invariant chain (Ii) complexes into a pathway that mediates the rapid and inefficient because specific proliferation occurred only with high (3 mg per ml) amounts of protein efficient generation and surface display of peptide-bound MHC class II. When we subjected used for pulsing. In contrast, at low (300-fold less) protein antigen-pulsing concentrations (10 µg immature, resting and TNF-a/IL-1b-exposed monocyte-derived (md)DC to metabolic labeling per ml) the DC stimulated an HEL-specific hybridoma very efficiently (175 pg IL-2 per ml), and anti-HLA-DR immunoprecipitation, we observed that class II ab-peptide complexes are suggesting that these DC may lack certain costimulatory molecules required for potent T cell formed more rapidly and efficiently in cytokine-activated than in nonactivated DC. Since complete stimulation. DC were very efficient at stimulating allogeneic T cells. When protein-antigen pulsed Ii degradation is a prequisite for peptide acquisition and surface export of MHC class II, we asked DC were injected into mouse footpads, priming could not be assessed because of an induced whether enhanced Ii-directed proteolytic activity may account for this increase in class IIab- autologous T cell response. We then injected hapten-modified DC subcutaneously and found that peptide formation by activated DC. Based on our observation that DC express the cysteine these mice exhibited significant contact sensitivity (CS) after hapten challenge 7 d later. To protease cathepsin (cat) S and that this molecule is involved in Ii degradation, we sought to determine whether these DC were the actual presenting cells, we used hapten-modified DC from interfere with the functionality of this enzyme in cytokine-activated and nonactivated mdDC. A or B mice and injected these cells into F1 hybrid (AxB), A or B mice and found that they Exposure of activated mdDC to LHVS, a specific active-site inhibitor of catS, delayed class II ab- induced significant CS in an MHC-restricted manner (p Ͻ 0.05). Hapten-modified-dead (heat peptide formation and export by 4–8 h and resulted in the appearance of a class II-associated, killed: 56 °C for 50 min) DC did not induce CS. Taken together, these studies indicate that these 13 kDa N-terminal Ii intermediate which contains lysosomal targeting motifs within its abluminal DC expanded from bone marrow may be lacking in certain costimulatory molecules important domain. Indeed, the LHVS-induced accumulation of the HLA-DR-associated Ii fragment for protein-antigen sensitization, but function very well in the induction of CS. Cells expanded correlated with the preferentional targeting/retention of MHC class II to/within lysosomal rather in this way may therefore not induce T helper cell responses for protein antigen sensitization, but than endosomal DC organelles. In contrast, the blockade of catS in nonactivated DC did not may be useful in helping to elucidate the afferent requirements of contact sensitization. appreciably affect the slower appearance kinetics of peptide-loaded MHC class IIab complexes in these cells. Active site-labeling experiments directly demonstrated that TNF-a/IL-1b signaling rapidly upregulates DC-bound catS activity. Thus, it appears that the regulation of catS activity in DC is subject to proinflammatory signals and, more importantly, that the dynamic regulation of catS activity allows DC to rapidly adapt the efficacy of its antigen processing and presentation apparatus to environmental danger signals. 524 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

007 008 Targeting Cultured Dendritic Cells to Peripheral Lymph Nodes Using a Platelet Bridge Flt3 Ligand Induces Skin Dendritic Cells and is Protective Against Disease Progression in C. Robert, G. Cheng, R. Fuhlbrigge, T. H. Wong, H. Falet, U. von Andrian and T. S. Kupper Susceptible BALB/C Mice Infected with Leishmania major Harvard Skin Disease Research Center, BWH, Boston, Massachusetts I. B. Kremer, K. D. Cooper,† and F. P. Heinzel*† Because of their unique and potent capacity to present antigens to naı¨ve T cells in peripheral Department of Dermatology and *Division of Geographic Medicine, Case Western Reserve lymph nodes (PLN), dendritic cells (DC) hold great promise as immunotherapeutic tools. DC- University, University Hospitals of Cleveland and †VA Medical Center, Cleveland, Ohio based therapies rely on the ability to grow large numbers of DC from CD34ϩ progenitors or Flt3 ligand (Flt3 L) is a hematopoietic growth factor that when administered to mice generates blood monocytes. Because naı¨ve T cells are most abundant in PLN, the capacity of cultured DC large numbers of dendritic cells (DC) in lymph nodes, liver and spleen. To investigate the effects to traffic to this tissue has been examined. When injected intracutaneously, cultured DC only of Flt3 L on DC in skin, we injected mice subcutaneously with human rFlt3 L (Elaine K. Thomas, migrate inefficiently to local draining lymph nodes. However, when injected intravenously, DC Immunex, Seattle; 10 µgor1µg daily for 10 d). Immunohistochemistry of skin biopsies harvested do not reach the PLN, but rather accumulate in extranodal sites. In PLN, the interaction of L- on day 11 demonstrated an increase in IaϩCD11bϩCD11cϩCD40ϩDC in the dermis of Flt3 L selectin with its counterreceptor PNAd, expressed on high endothelial venules (HEV), is the treated mice (6.7 Ϯ 0.36-fold increase; n ϭ 4). Three-color flow cytometry showed a dose- initial step in the extravasation of naı¨ve lymphocytes. We found that, regardless of progenitor cell dependent increase in CD11bϩCD11cϩCD40ϩCD3-CD19-cells in the LN of mice treated with or culture condition, cultured DC did not express L-selectin and could not tether or roll on Flt3 L (10 µg: 9.3 Ϯ 1.9-fold; n ϭ 4; 1 µg: 4.2 Ϯ 0.1-fold; n ϭ 2). LN cells of Flt3 L treated PNAd coated surfaces in a parallel plate flow chamber assay. It was recently shown (U.vA, 1997) mice showed a 20- Ϯ 2.3-fold increased IL-12 p40 production (5 ng per ml; n ϭ 2) compared that activated platelets (AP) could form a bridge between L selectin deficient T cells and HEV, with untreated mice. DC are highly efficient Ag-presenting cells, thus we hypothesize that the allowing these T cells to enter PLN independent of L-selectin. AP express P-selectin which can presence of large numbers of IL-12 producing DC in LN and skin will enhance immune responses bind to both T cell P-selectin-glycoprotein-ligand-1 (PSGL-1) and HEV PNAd. Because DC against infectious agents. Therefore, BALB/C mice, that are susceptible to infection with the express PSGL-1, we asked if they could bind (AP), and if this event could mediate DC-rolling intracellular parasite Leishmania major, were pretreated subcutaneously with Flt3 L for 10 d before on PNAd. By microscopic observation and two-color FACS analysis, we found that both DC infection with L. major in the hind footpads. Pretreatment of BALB/C mice with 10 µg Flt3 L readily bound labeled AP. Preincubation of cultured DC with AP conferred the capacity to roll was protective against disease progression as measured by a significantly reduced footpad swelling on PNAd under flow conditions. Microscopy confirmed that AP were bound to the rolling DC. (p ϭ 0.0002; n ϭ 3) compared with untreated L. major infected mice. The effectiveness of Flt3 L Because cultured DC constitutively express activated LFA-1, treatment of DC with AP may be is dose-dependent: pretreatment with 1 µg Flt3 L showed no protective effect. Protection was sufficient to mediate their extravsation from blood to LN. We propose that targeting of injected not observed when the mice were treated with Flt3 L starting on the day of infection even though DC to PLN using AP will improve the effectiveness of DC-based therapies. the Flt3 L treatment induced numbers of DC and IL-12 p40 production similar to mice that received Flt3 L only. In conclusion, intracutaneous Flt3 L pretreatment is an effective inducer of large numbers of IL-12 producing DC in LN and skin and induces protective immunity against L. major in normally susceptible mice.

009 010 The Upper Hair Follicle Contributes Young Transit Amplifying Cells to Newborn Mouse Engrailed-1 (En1) Expression by the Dermal Papilla Affects Hair Size in Mouse Skin Epidermis: Hypothesis of Bipotent Bulge Stem Cells M. B. Birge, C. -X. Tong, S. Mohan, J. Perone and C. A. Loomis G. Taylor, M. S. Lehrer, P. J. Jensen, T.-T. Sun,* and R. M. Lavker Ronald O. Perelman Department of Dermatology, Department of Cell Biology and Developmental Department of Dermatology, University of Pennsylvania, Philadelphia, Pennsylvania; *NYU, Genetics Program, Skirball Institute, NYU School of Medicine, New York New York, New York En1 is a homeobox containing transcription factor which is critical for normal embryonic The normally slow-cycling hair follicular epithelial stem cells located in the bulge zone give rise development. We have shown previously that En1 is required for formation of the embryonic to a new, lower follicle during each anagen. In addition, it has been postulated that the bulge limb signalling center, the apical ectodermal ridge (AER), as well as for specification of ventral stem cells participate in the maintenance of the epidermis. To test this hypothesis, we selectively mesenchyme and ectoderm fates on the distal limb. Postnatal En1 expression is important for tagged the transit amplifying (TA) cells of the upper follicle by a double-labeling technique, based normal volar ectoderm differentiation and pigmentation. Here we demonstrate that En1 expression on our observation that neonatal (4–7 d), follicular keratinocytes in the infundibular region cycle in the skin is not limited to volar ectoderm but is also expressed in dermal papillae. En1 is faster than epidermal keratinocytes (18 vs 24 h). Consistent with this observation, two consecutive expressed throughout the superficial mesenchyme of the back at µ14.5 d post coitum. Expression pulse labels, first with bromodeoxyuridine (BrdU) and then with tritiated thymidine (T), 18 h then spreads ventrally and acrally prior to the appearance of ectodermal hair buds. Expression apart, resulted in 32% of the BrdU-labeled infundibular cells but only 1.2% of the BrdU-labeled levels increase during late fetal development, but by the first day of life En1 expression is restricted epidermal cells, respectively, also being T (ϩ) and thus becoming double-labeled cells (DLC). to the papillary dermis and newly formed dermal papillae. Over the following 2 wk, expression Chases of 18 h and 36 h resulted in striking increases in epidermal DLC to 18% and 39%, in the papillary dermis is extinguished and expression of En1 in the mesenchyme becomes respectively. Moreover, most of these epidermal DLC were near follicular orifices. These results restricted to the dermal papillae cells. As the hair follicles move into the first catagen-anagen stage strongly indicate that follicular DLC can emigrate to the epidermis. Although based on clinical En1 expression appears to be maintained throughout the hair cycle, although at low levels. Analysis observations of burn patients it has frequently been said that follicular cells can repair the entire of hairs from mutant mice indicates that they are much thinner than wild type hairs. This epidermis, our results provide the first experimental evidence that follicular infundibulum observation is consistent with the known role of dermal papillae cells in regulating the size of the contributes proliferating cells to normal neonatal epidermis. Furthermore, our data support the follicle and keratinized hair shaft, and suggests that En1 plays a critical role in regulating this function. idea that follicular epithelial stem cells are bipotent, in that they are not only responsible for generating a lower follicle, but also contribute proliferating cells to neonatal epidermis. The double-labeled infundibular cells most likely represent the relatively young TA1 cells in the scheme of bulge stem cells ® infundibular TA1 cells ® epidermal TA2 cells which can continue to proliferate and thus may contribute to epidermal expansion, repair, and homeostasis.

011 012 WNT Signaling in the Control of Hair Growth and Structure Patterns of Hairless (hr) Gene Expression in Mammalian Skin S. E. Millar, K. Willert, P. C. Salinas, H. Roelink, R. Nusse, D. J. Sussman and G. S. Barsh A. M. Christiano,*† J. P. Sundberg,‡ W. Ahmad,* and A. A. Panteleyev* Department of Dermatology, University of Pennsylvania, Philadelphia, Pennsylvania; Howard *Department of Dermatology and †Genetics and Development, Columbia University, New York, Hughes Medical Institute, Stanford University Medical Center, Stanford, California; Department New York; ‡The Jackson Laboratory, Bar Harbor, Maine of Developmental Biology, Stanford University, Stanford, California; Departments of Pediatrics The hairless (hr) gene encodes a putative single zinc-finger transcription factor with as yet and Genetics, Stanford University, Stanford, California; and Division of Human Genetics, incompletely understood functions. Mutations in the h gene cause severe hair follicle (HF) University of Maryland, Baltimore, Maryland dysfunction, resulting in generalized atrichia in humans (MIM 209500) and complete shedding of Characterization of the molecular pathways controlling differentiation and proliferation in hairs in mice. In order to further explore the role of the h gene in skin and HF biology, we mammalian hair follicles is central to our understanding of the regulation of normal hair growth, studied h mRNA localization in mouse and human skin by nonradioactive in situ hybridization the basis of hereditary hair loss diseases, and the origin of follicle-based tumors. We have found with digoxigenin-labeled riboprobes during defined stages of HF morphogenesis and hair cycling. that the proto-oncogene Wnt3, which encodes a secreted paracrine signaling molecule, is expressed In the interfollicular epidermis, h mRNA immunoreactivity was consistently localized to the in developing and mature hair follicles and that its over-expression in transgenic mouse skin causes suprabasal spinous cell layer and gradually declined along with keratinocyte differentiation. The a short hair phenotype due to altered differentiation of hair shaft precursor cells, and cyclical basal and highly differentiated keratinocytes of the granular layer were h mRNA-negative. During balding resulting from hair shaft structural defects and associated with an abnormal profile of the early stages of HF morphogenesis, h mRNA was detected over the inner keratinocyte protein expression in the hair shaft. A putative effector molecule for WNT3 signaling, the compartment of downward growing hair peg. With the initiation of hair shaft development, h cytoplasmic protein Dishevelled 2 (DVL2), is normally present at high levels in a subset of cells mRNA became concentrated in the infundibulum, in the hair matrix, and in the inner root sheath in the outer root sheath, and in precursor cells of the hair shaft cortex and cuticle which lie (IRS) cone, while the dermal papilla and outer root sheath were always h mRNA-negative. In immediately adjacent to Wnt3 expressing cells. Over-expression of Dvl2 in the outer root sheath the HF infundibulum, like in the interfollicular epidermis, h gene expression remained constant, mimics the short hair phenotype produced by over-expression of Wnt3, supporting the hypothesis while in the proximal portion of the HF, it is subject to hair cycle-dependent changes. During that Wnt3 and Dvl2 have the potential to act in the same pathway in the regulation of hair the middle stages of HF morphogenesis and the anagen stage of the hair cycle, h mRNA was growth. These experiments demonstrate a role for WNT signaling in the control of hair growth detected in the proximal hair matrix and the IRS. With the onset of catagen HF transformation, h and structure, as well as presenting the first example of a mammalian phenotype resulting from gene expression gradually declined in the regressing IRS; shortly but dramatically increased in the over-expression of a Dvl gene and providing an accessible in vivo system for analysis of mammalian developing club hair; and is prominently upregulated in the epithelial cells adjacent to the DP WNT signaling pathways. during the final stages of its upward movement. The colocalization of h gene expression with the sites of the morphological defects in hairless skin implicate the h gene as a key factor in regulating the basic cellular processes during hair follicle catagen, including club hair formation, maintenance of dermal papilla-epithelial integrity, inner root sheath disintegration, and keratinocyte apoptosis in hair matrix. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 525

013 014 Activation of Hedgehog Target Gene Expression in Basal Cell Carcinomas Sonic Hedgehog Opposes Epithelial Cell Cycle Arrest J. Bonifas, S. Pennypacker, E. H. Epstein, Jr H. Fan, S. A. Wee and P. A. Khavari Department of Dermatology, University of California San Francisco, San Francisco, California VA Palo Alto and Stanford University, Stanford, California Mutations in the hedgehog signaling pathway underlie the abnormal behavior of basal cell Stratified epithelium displays an equilibrium between proliferation and cell cycle arrest, a balance carcinomas, but the mechanism by which these signaling aberrations causes the phenotypic that is disrupted in basal cell carcinoma (BCC). Sonic hedgehog (Shh) pathway activation appears abnormalities remains to be elucidated. Therefore, we have assessed these tumors by ribonuclease sufficient to induce BCC, however, the way it does so is unknown. To determine if Shh impacts protection for expression of genes whose activation in model systems is controlled by hedgehog epithelial cell cycle regulation, we expressed Shh in human epidermis regenerated on SCID mice signaling, and of genes that encode hemidesmosomal proteins, which characteristically are lost in from retrovirally transduced keratinocytes in an approach that effects Shh pathway activation. basal cell carcinomas. In addition, to identify what WNT family members are expressed in normal Shh[ ϩ ] epidermis is hyperplastic with Ͼ6-fold increase in cell numbers/100 µM surface length keratinocytes and basal cell carcinomas, mRNA was reverse transcribed and amplified with primers by 3 wk vs unengineered, GFP[ ϩ ] and lacZ[ ϩ ] controls. This is accompanied by that anneal to consensus WNT family sequences. Amplified product was cloned and inserts were hyperproliferation, with Ͼ10-fold increase in both Ki-67 expression and BrdU incorporation by sequenced. As controls for the amount of mRNA content, we used three housekeeping genes – Shh[ ϩ ] epidermis. In contrast to normal, Ki-67 and BrdU[ ϩ ] cells are seen well above the GAPDH, ACTIN, and CYCLOPHILIN, the relative levels were consistent in each sample. As basal layer in Shh[ ϩ ] epidermis, suggesting a failure to undergo the cell cycle arrest that normally reported previously all BCC have activation of PATCHED and GLI1 message. GLI2, BMP2, and occurs prior to outward migration. To determine if Shh confers resistance to differentiation- BMP4 levels were also increased and varied over an µ 6-fold range. We assessed mRNA levels associated cell cycle arrest, cell cycle distribution was analyzed in early passage Shh[ ϩ ] and of the four major known components of heidesmosomes-a6 and b4 INTEGRINS and BPAG1 control cells in vitro after calcium stimuli and, in a separate series of experiments, after high and 2. Of these the most consistent abnormalities were detected in b4 INTEGRIN. Compared efficiency retroviral transduction with a vector for the CDK inhibitor p21CIP1. Shh-expressing to normal keratinocytes all BCC had at least 90% reduction of expression. The a6 message was cells fail to exit S and G2/M in response to calcium-induced differentiation and also resist p21CIP1 decreased by 90% in seven of eight tumors. One BCC had normal levels. However, both in induced growth arrest. To test if Shh augments the long-term growth capacity of nonimmortalized RNA extracted from skin and suction blister epidermis only trace amounts of a6 and b4 message epithelial cells, we performed serial passaging experiments. Shh[ ϩ ] cells resist replicative were detected. In contrast, similar amounts of message for BPAG2 were obtained in keratinocytes, exhaustion, generating a final cumulative cell yield 5–17-fold greater than controls. Enhanced skin and epidermis. Levels of BPAG2 were variable, ranging from near complete loss to some growth of Shh[ ϩ ] cells over GFP[ ϩ ] and lacZ[ ϩ ] controls first becomes evident at passage increase. Most tumors showed an increase of BPAG1. We detected at least six different WNT 8. While controls cease proliferating by passage 13, Shh[ ϩ ] cells proliferate actively until passage family members expressed in keratinocytes and/or BCC, of which WNT7b was found most 16. These data indicate that Shh enhances the replicative capacity of normal epithelial cells and commonly. Probes specific for WNT1, 5a, 7b, and 13 were constructed and mRNA levels were confers resistance to physiologic triggers of keratinocyte cell cycle arrest, suggesting a dual growth assessed. Normal keratinocytes had very low levels barely detectable after a week of exposure. By de-regulation mechanism for Shh-induced neoplasia. contrast, mRNA from these WNT family members was easily detected in some BCC. In conclusion, we have found clearly increased levels of mRNA not only for PATCHED but also for multiple WNT, GLI, and BMP family members, and a loss of hemidesmosmal gene expression, suggesting that these genes too may be under hedgehog control, at least indirectly.

015 016 Regulation of Shh Target Gene Expression Plays a Critical Role in Hair Follicle and BCC Formation Cyclopamine Inhibits Sonic Hedgehog Signaling and Blocks Morphogenesis of Vibrissa Follicle A. E. Oro, K. Higgins and M. P. Scott Explants Department of Dermatology and Dev Biology, HHMI, Stanford University, Stanford, California R. Z. Swan, M. Grachtchouk, E. K. Robertson, M. K. Cooper, P. A. Beachy and A. A. Dlugosz Activation of hedgehog (Hh) target genes plays a central role in both the formation of normal Department of Dermatology and Comprehensive Cancer Center, University of Michigan, Ann hair and basal cell carcinomas (BCC), although how the latter are related is unclear. Using a Arbor, Michigan; Howard Hughes Medical Institute, Johns Hopkins University, Baltimore, mouse containing a patched(ptc1)-lacz reporter gene, we find ptc1 expression occurs only in the Maryland growing hair epithelium and dermal papilla. Examination of the skin of adult patched (ptc) Identification of pharmacological inhibitors of the Shh pathway could provide a novel approach heterozygous mice reveals ectopic activation of ptc1 and microscopic epithelial invaginations in for defining the normal functions of this pathway in skin and other organs, and may ultimately mice older than 9 mo but not in wild type littermates. These invaginations differentiate into hair be useful therapeutically given the association of constitutively activated Shh signaling and basal follicle epithelium rather than continuing to grow into large BCC. The invaginations arise from cell carcinoma. We selected cyclopamine for analysis since exposure of laboratory animals to this interfollicular skin and bulge regions of the hair suggesting that follicular and interfollicular drug in utero elicits a spectrum of developmental defects strikingly similar to those seen in Shh–/– keratinocytes are capable of hair differentiation. To assess the distribution of interfollicular cells mice. Treatment of cultured embryonic dermal cells with recombinant Shh resulted in a dramatic capable of differentiating toward hair, we have ectopically expressed gli-1, a mediator of the stimulation of the Shh target genes Gli1 and Ptc1. This response was inhibited by cyclopamine, transcriptional effects of Shh. These results suggest that many adult keratinocytes outside the bulge indicating that this drug is capable of blocking exogenous activation of the Shh pathway in are capable of differentiating into hair follicle epithelium and that the temporal and spatial monolayer cultures of cutaneous mesenchymal cells. We next assessed the effects of cyclopamine regulation of Shh target gene expression plays a critical role in hair follicle and BCC formation. on vibrissa follicle development using tissue explants from mouse embryos at 13.5 d of gestation, shortly after the initiation of vibrissa formation. Over a 4–8-d period in culture, control explants develop normal-appearing vibrissa follicles with well-formed dermal papillae, inner and outer root sheaths, and hair shafts. Cyclopamine blocked this robust morphogenetic program in a dose- dependent manner. A neutralizing Shh antibody, but not control IgG, also inhibited vibrissa follicle development in explant cultures. Expression of Gli1 mRNA was markedly reduced in explants treated with cyclopamine, providing strong evidence that this agent effectively blocks endogenous activation of the Shh pathway in a complex tissue environment. Despite the nearly complete inhibition of follicle morphogenesis, histological analysis revealed that cyclopamine-treated explants produced short, keratinized hair shaft-like structures, and accumulated mRNA encoding murine hair keratin A1 and the hair cortex marker Hacl-1. These results are remarkably similar to those described in pellage follicles of Shh–/– skin grafts on nude mice, which produced abortive hair shafts expressing hair-specific keratin despite a profound inhibition of morphogenesis. Our findings strongly suggest that Shh is operating as an inductive morphogenetic signal that is required during early stages of vibrissa follicle development, and establish the utility of cyclopamine as a novel tool for inhibiting Shh signaling in skin.

017 018 Interleukin 12 Prevents Apoptosis of Dendritic Cells by Downregulation of Fas and Induction of Clusterin Mediates Resistance To Apoptotic Cell Death Induced by Oxidative Stress and Antiapoptotic Proteins Heat Shock A. Schwarz, K. Grosse-Heitmeyer, T. Brzoska, H. Kalden, S. Grabbe, T. Luger and T. Schwarz I. Viard, P. Welirli, L. Jornot,* R. Bullani, J. L. Vechietti, J. A. Schifferli,† J. Tschopp,‡ and L. Ludwig Boltzmann Institute for Cell Biology and Immunobiology, Department of Dermatology, E. French Mu¨nster, Germany Departments of Dermatology (DHURDV) and *Pneumology, Geneva University, Geneva, Irradiation of mice with ultraviolet light (UV) prior to hapten sensitization induces immune Switzerland; †Department of Medicine, Basel University, Basel, Switzerland; and ‡Institute of tolerance which is mediated via T suppressor cells. Recently it was shown that coincubation of Biochemistry, Lausanne University, Lausanne, Switzerland dendritic cells (DC) with T cells from tolerized mice results in apoptosis of DC. DC death is Clusterin is a secreted glycoprotein that is strongly expressed in tissues regressing as a consequence mediated via the apoptosis related Fas/Fas-ligand system and can be prevented by addition of of enhanced apoptotic cell death. In in vitro models of apoptosis, clusterin expression is strictly interleukin-12 (IL-12). Since IL-12 can also break UV-mediated immune tolerance in vivo, we confined to surviving cells, suggesting that it is involved in cell survival rather than death. To were interested to study the mechanisms by which IL-12 rescues DC from apoptosis. Incubation determine the role of clusterin in keratinocyte biology, we have analyzed its expression and of the murine DC line XS52 with recombinant Fas ligand induced apoptosis which was significantly function in response to relevant cellular stimuli, including heat shock and oxidative stress. We reduced upon preincubating cells with IL-12. FACS analysis revealed that IL-12 significantly show that in primary keratinocytes, A431 and HaCaT cells, both a transient heat shock, and reduced constitutive and interferon-g induced Fas surface expression. Downregulation of Fas by various oxidative stresses (hydrogen peroxide, superoxide anion, hyperoxia and UVA exposure) IL-12 was also confirmed by northern blot analysis. Since IL-12 did not completely suppress Fas induce a rapid and strong increase in clusterin gene transcription, protein synthesis and secretion, expression, we hypothesized that other pathways might also be involved in the antiapoptotic thus resulting in increased extracellular levels of the clusterin. To investigate the function of activity of IL-12. Therefore, the effect of IL-12 on the expression of the antiapoptotic proteins clusterin, antisense-transfectants were generated in A431 cells. Several antisense-transfected A431 FLIP (FLICE inhibitory protein) and bcl-xL was studied. FACS analysis of permeabilized cells clones were selected and shown to express virtually no clusterin at the mRNA and protein level using an anti-FLIP antiserum revealed modulation of FLIP expression upon stimulation with Fas when compared to mock-transfected or wild-type A431 cells. When exposed to heat shock or ligand. Exposure of cells to IL-12 significantly upregulated expression of FLIP. Induction of FLIP various oxidative stresses (hydrogen peroxide, UVA, diethymaleate) under conditions that induce by IL-12 was also confirmed by RT-PCR. In addition, IL-12 significantly induced bcl-xL. Taken apoptosis, antisense transfectants were shown to be far more sensitive to apoptotic cell death than together, IL-12 seems to exert its antiapoptotic capacity by downregulating Fas and by inducing mock-transfected and wild type A431 cells. No difference in susceptibility to apoptosis was observed the antiapoptotic proteins FLIP and bcl-xL. however, when these cells were exposed to pKC-mediated pro-apoptotic stimuli staurosporine. Taken together, our results show that clusterin gene expression is selectively and strongly induced in response to heat shock and oxidative stress in keratinocytes, and that it confers significant cellular protection against apoptosis induced by heat shock and oxidative stress. 526 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

019 020 Importance of NF-kB in Regulating Keratinocyte Apoptosis NF-kB Function Determines Localization and Features of Programmed Cell Death In Epidermis J. Z. Qin, J. Panella, V. Chaturvedi, M. F. Denning and B. J. Nickoloff C. S. Seitz, R. A. Freiberg and P. A. Khavari Department of Pathology, Loyola University, Chicago, Illinois Stanford University, Stanford, California While NF-kB can influence transcription of numerous and diverse signal transduction pathways Specialized forms of programmed cell death lacking characteristic morphologic features of apoptosis and cytokine-mediated cellular responses, we explored its role in UVB-induced apoptosis. occur in terminally differentiating tissues, including the outer layers of stratified epithelium. NF- Proliferating KC and an immortalized cell line (HaCaT) are highly susceptible to UVB induced kB subunits, previously shown to inhibit apoptotic cell death in lymphoid and mesenchymal cells, apoptosis. However, if normal human KC are pretreated for 24 h with IFN-g plus TPA, they translocate from the cytoplasm to the nucleus in differentiating layers of epidermis, raising the acquire a near-complete resistance to UVB induced apoptosis. Whereas similarly treated HaCat possibility that NF-kB may regulate terminal differentiation-associated cell death. To test this cells do not acquire this protective phenotype, even though HaCaT cells have a comparable possibility, we studied the impact of altering NF-kB function on epithelial cell death in vitro and degree of growth arrest (GA) by IFN-g plus TPA. Since NF-kB can activate transcription of genes in vivo. High titer amphotropic retroviral expression vectors for NF-kB subunits p50 and p65 encoding for several cell survival proteins, NF-kB localization and function in KC and HaCaT were used to activate and the IkBaM super-repressor (which blocks all five mammalian NF-kB cells was characterized under conditions where they are sensitive or resistant to apoptosis. subunits) was used to block NF-kB function in primary human keratinocytes. Serial transduction Immunostaining and western blot analysis of isolated nuclei revealed that KC had weak but with these vectors and a retroviral vector for human Fas/CD95 was used to study NF-kB effects detectable levels of p50, p65, or Rel b; and IFN-g plus TPA enhanced nuclear levels of on Fas triggered apoptosis. In vitro, NF-kB activation protects keratinocytes from apoptosis induced these proteins within 8 h. This nuclear translocation of NF-kB was preceded by cytoplasmic by Fas with 5.4-fold less cells undergoing TUNEL[ ϩ ] apoptosis at 7 h after triggering with the phosphorylation and degradation of Ik-Ba. Electrophoretic gel shift analysis, and supershift assays anti-Fas antibody (8.5% vs 45.9% of cells). Conversely, inhibition of endogenous NF-kB subunits confirmed significant and rapid (4–8 h) induction of NF-kB binding following exposure to IFN- by IkBaM renders cells 43.5% more susceptible to Fas-triggered cell death than control. Consistent g plus TPA. By contrast, HaCaT cells displayed constitutive nuclear levels of p50, p65, and Rel with this, NF-kB blockade leads to a 7.2-fold increase in apoptotic cells triggered by TNFa over b, but there was no change in either IkBa or NF-kB in HaCaT observed after IFN-g plus TPA, normal control, indicating that endogenous NF-kB subunits protect against cell death in epithelial by several different assays. To further explore NF-kB induced cell survival pathways, RNAse cells. Transgenic mice expressing IkBaM in epidermis and displaying blockade of NF-kB nuclear protection assays for inhibitors of apoptosis were performed on proliferating KC before and after translocation in vivo exhibit regions of spontaneous premature cell death in the spinous and GA induced by IFN-g plus TPA. Untreated proliferating KC had either low or undetectable granular layers. This premature death is characterized by classic histologic features of apoptosis and levels of xIAP, TRAF1, TRAF2, c-IAP1, c-IAP2, and IEX–1 L mRNA but after IFN-g plus evidence of DNA strand breakage with Ͼ 15-fold induction of TUNEL[ ϩ ] cells over normal TPA, there was strong induction of all these transcripts. HaCaT cells constitutively expressed littermates. These data indicate that NF-kB inhibits apoptosis in keratinocytes and prevents a relatively low or undetectable levels of these transcripts, but IFN-g plus TPA did not enhance the default apoptotic pathway in epithelial cells preparing to undergo more specialized forms of expression of either xIAP, TRAF-1, TRAF-2, c-IAP1, or IEX–1 L. Induction of these KC programmed cell death. transcripts after IFN-g plus TPA was also associated with prevention of caspase 8 activation and inhibition of UVB induced apoptosis. We conclude that IFN-g plus TPA activate NF-kB in normal KC inducing several cell survival proteins protective against UVB induced apoptosis. HaCaT cells do not increase their NF-kB translocation after GA, with subsequent failure to upregulate a cell survival pathway necessary to resist UVB induced apoptosis.

021 022 Targeted Expression of c-myc in the Epidermis Alters Normal Proliferation, Differentiation and A Three Dimensional Culture System for Studying Melanoma Progression UV-B Induced Apoptosis Y. Shellman, J. P. Gendall, I. Maxwell and D. A. Norris R. L. Waikel,* X. -J. Wang,*† and D. R. Roop*† Department of Dermatology, University of Colorado Health Science Center, Denver, Colorado Departments of *Cell Biology and †Dermatology, Baylor College of Medicine, Houston, Texas A crucial phase of melanoma progression is the switch from radial growth phase (RGP) to invasive c-Myc overexpression has been associated with several types of human cancers. To study the role vertical growth phase (VGP) melanoma. We propose that a three dimensional (spheroid) anchorage- of c-myc in epidermal differentiation and carcinogenesis, a transgenic mouse model was created independent tissue culture system on agar is superior for modeling in vitro VGP melanoma growth to overexpress c-Myc in the epidermis. Human c-myc 2 cDNA was subcloned into a 6.5-kb and survival since VGP melanoma invades away from anchorage at the dermal epidermal junction, mouse loricrin expression vector, ML.myc2. This loricrin promoter primarily directs expression and from anchorage-dependent signals there. Our purpose was to demonstrate differences in in the epidermis in both proliferating and differentiated keratinocytes. On day 4, ML.myc2 apoptosis susceptibility in RGP vs VGP melanoma cell lines grown in spheroid culture. We have transgenic pups develop a hyperkeratotic phenotype, which progressively worsens until day 7. previously shown that stable transfection of plasmids expressing activated H-or N-ras into the low Upon histological analysis, both hyperplasia and hyperkeratosis were evident. Bromodeoxyuridine invasive potential melanoma (RGP)WM35, produced cell lines with characteristics typical of the (BrdU) incorporation revealed that transgenic mice had a 3-fold increase in the number of VGP of melanoma. In this study, parent, ras transfected, and plasmid control cell lines were also proliferating cells as compared with a normal littermate. Proliferative cells in the ML.myc2 examined in vitro for susceptibility to apoptosis and antiapoptotic defenses. In monolayer adherent epidermis were also found to be suprabasal, suggesting an inhibition of terminal differentiation in cultures, all cell lines expressed high levels of the antiapoptotic proteins Bcl-2, Bcl-xL and Mcl-1. keratinocytes. Inhibition of terminal differentiation by c-Myc overexpression was further suggested However, in spheroid culture, the WM35 parent and control cell lines showed highly significant by aberrant expression of differentiation markers, keratin 1, keratin 6, loricrin, and filaggrin in decreases in expression of Bcl-2, while the ras transfected cells maintained high levels of all three ML.myc2 transgenic mice. Interestingly, ML.myc2 keratinocytes exhibited resistance to UV-B antiapoptotic. Differences in susceptibility to apoptosis correlated inversely with the levels of Bcl- induced apoptosis both in vitro and in vivo. These findings provide evidence that overexpression 2 expression. These data show that there is differential control of expression of the antiapoptotic of c-Myc in the epidermis induces proliferation, inhibits terminal differentiation and decreases the proteins Bcl-2, Bcl-xL and Mcl-1 in melanomas. Growth of melanomas on tissue culture plastic sensitivity of keratinocytes to UV-B induced apoptosis. maintains expression of Bcl-2, while anchorage independent growth in spheroid culture does not. We hypothesize that activated ras plays an important role in melanoma progression from RGP to VGP by providing a survival advantage for melanoma cells in the dermal environment, away from epidermal-derived growth factors and from attachment to the dermal-epidermal junction. Activated ras would promote melanoma survival and invasion in the dermis by maintaining expression of the full component of antiapoptotic proteins, especially Bcl-2.

023 024 Repeated Exposure of Ultraviolet Light Induces Resistance to UV-Induced Apoptosis in a Human Defective Death Signaling by Fas and TRAIL-R1 in Malignant Melanoma Melanoma Cell Line through Downregulation of CD95 R. Bullani, P. Wehrli, I. Viard, J. -H. Saurat, J. Tschopp,* and L. E. French Y. Aragane, A. Maeda, A. Amatsu, T. Tezuka and T. Schwarz* Department of Dermatology (DHURDV), Geneva University Hospital, Geneva, Swizerland; and Department of Dermatology Kinki University School of Medicine, Osaka, Japan; *Department *Institute of Biochemistry, Lausanne University, Lausanne, Switzerland of Dermatology, University of Mu¨nster, Mu¨nster, Germany Death receptors detect the presence of extracellular death signals and can rapidly trigger apoptotic Ultraviolet irradiation (UV) induces apoptosis in a variety of cell types. Studying the effect of UV cell death. They are crucial regulators of cell numbers and may contribute to melanoma on melanoma cells we observed that repeated UV exposure renders cells of the human melanoma development through defective signaling. To date six human death receptors (Fas, TNF-R1, cell line G361 resistant to UV-induced apoptosis. Compared to normal G361 cells, repeatedly UV TRAMP [wsl/Apo-3/DR-3], TRAIL-R1 [DR-4], TRAIL-R2 [DR-5], and DR-6) have been exposed G361 (ruvexG361) did not undergo apoptosis following a single UV exposure as identiﬁed, and signaling by these can be inhibited by cellular FLIP (FLICE [Caspase-8]-inhibitory demonstrated by DNA laddering and cleavage of the death substrate poly(ADP)ribose polymerase. protein). Expression of three death receptors (Fas, TRAIL-R1, TRAIL-R2) and their common Resistance was associated with downregulation of apoptosis related surface molecule CD95 (Fas/ inhibitor FLIP was analyzed by RNase protection, FACS and/or western blot analysis in 13 Apo-1) which recently was shown to be involved in UV-induced apoptosis. CD95 was melanoma cell lines, and by immunohistochemistry in melanocytic lesions. Signaling by these downregulated in ruvexG361 cells both at protein and at mRNA levels as demonstrated by three death receptors was determined in primary melanocytes and melanoma cell lines by a cell western blot analysis and RT-PCR. Accordingly, ruvexG361 cells were resistant to apoptosis death assay using recombinant FasL and TRAIL in vitro. In melanoma cell lines, frequent loss of induced by CD95 ligand. Transfection of ruvexG361 cells with an expression vector encoding Fas (40% of cases) and TRAIL-R1 (70% of cases) expression was observed. In all lines, expression CD95 restored the apoptotic sensitivity both to CD95 ligand and UV. Together, these data of TRAIL-R2 was strong, and that of FLIP was weak to moderate. In tissue sections of malignant demonstrate that CD95 plays an important role during UV-mediated apoptosis and secondly imply melanomas, loss of Fas (95% of cases) and induction of FLIP (70% of cases) expression were that repeatedly UV-exposed melanoma cells might escape elimination by the CD95/CD95 L frequently observed. Functional assays of the sensitivity of melanoma cell lines to recombinant system in vivo through downregulation of CD95. In addition, this may be suitable model allowing FasL and TRAIL in vitro (cell death assay) showed that 55% of the lines tested were resistant to to elucidate the mechanisms involved in negative regulation of CD95. both FasL and TRAIL. In the vast majority of cases, resistance to FasL and TRAIL was due to loss of Fas and TRAIL-R1 expression, respectively. None of the resistant cases could be attributed to selective modulation of TRAIL-R2, TRAIL-R3 (DcR1), FLIP, FADD, or Caspase-8 expression. Taken together, our results show that loss of Fas and TRAIL-R1 expression is frequent in melanomas and results in resistance to FasL-and TRAIL-mediated cell death, thereby potentially conferring a growth advantage to the tumor. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 527

025 026 Expression and Functional Characterization of the Largest Collagenous Domain of Human BPAG2 (Type XVII Collagen) AsSOCIATES with the N-Terminal Domain of BPAG1 in Collagen XVII/BP180 Hemidesmosomal Plaques K. Tasanen, J. Eble,* M. Aumailley,† H. Schumann,‡ J. Baetke,‡ and L. Bruckner-Tuderman‡ S. B. Hopkinson and J. C. R. Jones Department of Dermatology, University of Oulu, Oulu, Finland; *Department of Physiological Department of Cell and Molecular Biology, North-western University Medical School, Chemistry, University of Mu¨nster, Mu¨nster, Germany, †Department of Biochemistry, University Chicago, Illinois of Cologne, Cologne, Germany; ‡Department of Dermatology University of Mu¨nster, Mu¨nster, The hemidesmosome is a multimolecular junction in those epidermal cells that lie directly on the Germany basement membrane, and acts to tether the keratin-containing intermediate filaments of the Collagen XVII acts as an autoantigen in autoimmune blistering skin diseases and is a target for cytoskeleton to the cell surface. A major component of the hemidesmosome plaque is the 230 kDa mutations in junctional epidermolysis bullosa. Low levels of expression in tissues have impeded bullous pemphigoid autoantigen (BPAG1). BPAG1 binds directly to the keratin cytoskeleton via the functional studies of specific domains. Therefore recombinant Col15, the largest collagenous its C-terminus. A second bullous pemphigoid antigen of 180 kDa (BPAG2, type XVII collagen) domain of human collagen XVII, was expressed in 293 EBNA cells. This fragment of 241 amino is a type II transmembrane component of the hemidesmosome, whose N-terminus, like BPAG1, acids, designated as ERF-ColXVII-Col15, was purified by DEAE-cellulose and FPLC-MONO- is located in the electron dense cytoplasmic inner hemidesmosomal plaque with which keratin S chromatography. It was sensitive to collagenase digestion and recognized by a specific antibody bundles associate. Using yeast two-hybrid technology as well as recombinant protein fragments, spanning the same domain. Trypsin digestions and circular dichroism spectra as probes for protein we show that an N-terminal fragment of BPAG1 can bind directly to the cytoplasmic domain of folding indicated that ERF-ColXVII-Col15 has triple-helical conformation with a melting BPAG2. Furthermore, deletion analyses identify a region of 424 amino acids within the cytoplasmic temperature at 23°C and that the native comformation is strongly dependent on the presence of domain of BPAG2 that is crucial for such an interaction. Our results support the view that, at the ascorbic acid in the culture medium. Epitope mapping studies showed that ERF-ColXVII-Col15 site of hemidesmosomes, BPAG2 provides a membrane anchor for the keratin cytoskeleton via its was recognized by autoantisera of both bullous pemphigoid and linear IgA dermatosis patients. interaction with BPAG1. Interestingly, ERF-ColXVII-Col15 promoted cell adhesion of A431, Wi26, and L132 cells by a b1 integrin mediated mechanism. In conclusion, our studies indicated that ascorbic acid is absolutely required for the production of correctly folded collagen XVII and that the Col15 domain has a function in cell adhesion. Immunological targeting of this domain contributes to dermal–epidermal separation in blistering skin diseases.

027 028 Phosphorylation Reversibly Regulates the Association of Human Keratinocyte Cell–Cell Junc- The Head Domain of Plakophilin-1 Binds to the Amino-Terminal Domain of Desmoplakin and tion Proteins Enhances Desmoplakin Recruitment to Desmosomes: Implications for Cutaneous Disease D. S. Rubenstein, P. Hu and E. J. O’Keefe A. P. Kowalczyk, M. Hatzfeld, E. A. Bornslaeger, D. S. Kopp, J. E. Borgwardt, C. M. Corcoran, Department of Dermatology, UNC-CH, Chapel Hill, North Carolina A. Settler and K. J. Green To investigate how keratinocytes regulate cell–cell adhesion, we examined by confocal microscopy Departments of Pathology and Dermatology, North-western University Medical School, Chicago, and immunoprecipitation tyrosine phosphorylation of junction proteins in cultured human ker- Illinois; and The Molecular Biology Group of the Medical Faculty, Halle, Germany atinocytes. The contribution of desmosomes to epidermal integrity is evident in the inherited blistering Tyrosine phosphorylation produced by incubation of keratinocytes with the tyrosine phosphatase disorder associated with the absence of a functional gene for plakophilin-1 (PKP-1). To deﬁne inhibitor peroxivanadate resulted in (i) rapid loss of cell–cell adhesion, (ii) redistribution of the function of PKP-1 in desmosome assembly, interactions between the desmosomal cadherins, plakoglobin and b-catenin, (iii) loss of a-catenin staining, and (iv) tyrosine phosphorylation of desmoplakin, and the armadillo (arm) gene family members plakoglobin and PKP-1 were examined. plakoglobin and b-catenin. In keratinocytes treated with tyrosine phosphatase inhibitors, plakoglo- In transient expression assays, PKP-1 dramatically enhanced the recruitment of the desmoplakin bin-a-catenin binding was reduced, but could be restored by removal of phosphate from plakoglobin amino-terminal domain to intercellular junctions and formed complexes with desmoplakin that with phosphatase. b-catenin-a-catenin binding was increased in the presence of phosphatase. could be coimmunoprecipitated. Furthermore, the nonarm head domain of PKP-1 enhanced Additionally, tyrosine phosphorylation of b-catenin and plakoglobin reversibly decreased their desmoplakin recruitment to junctions, and this domain of PKP-1 also bound directly to the association with E-cadherin. desmoplakin amino-terminal domain in yeast two hybrid assays. In contrast to the arm domain of Thus, in human keratinocytes (i) tyrosine phosphorylation decreased the association of adherens PKP-1, the arm domain of plakoglobin did bind to desmoplakin, suggesting that the arm repeats junction proteins, whereas removal of phosphate from tyrosine promoted their association, and of these two related junctional proteins perform different functions. It is proposed that plakoglobin (ii) tyrosine phosphorylation of plakoglobin and b-catenin had a direct and reversible role in functions as a linker between the cadherins and desmoplakin and that PKP-1 enhances lateral regulating the cadherin-plakoglobin-a-catenin and cadherin-b-catenin-a-catenin complexes, interactions between desmoplakin molecules. These results indicate that PKP-1 plays an important respectively. role in desmoplakin recruitment to the desmosome and provide a mechanistic explanation for the epidermal blistering disorder that has been described in patients lacking PKP-1.

029 030 Differential Effects of Dominant Negative Mutants of Desmoglein, Desmocollin, and E-cadherin Stoichiometric Differences Among Cadherin/Catenin Complexes: Implications for Isoform Specific on Cell–Cell Adhesion of Keratinocytes Functions of Desmosomal Cadherins Y. Hanakawa, M. Amagai, Y. Shirakata, K. Sayama and K. Hashimoto L. J. Bannon and K. J. Green Department of Dermatology, Ehime University, Ehime; Department of Dermatology, Keio Departments of Pathology and Dermatology, North-western University Medical School, University, Tokyo, Japan Chicago, Illinois Epithelial cell–cell adhesion is considered to be important in development and maintenance of Cadherins are calcium dependent cell–cell adhesion molecules that are expressed in a variety of tissue integrity of stratified squamous epithelium. The purpose of this study is to investigate the cell and tissue types. In epithelial cells, a subclass of cadherins termed the desmogleins and differential roles of classic and desmosomal cadherins in the cell–cell adhesion of keratinocytes. desmocollins are found clustered in intercellular junctions known as desmosomes. These desmosomal We constructed recombinant adenoviruses (Ad) containing truncated mutants of E-cadherin (E- cadherins are expressed as three different isoforms (Dsg1–3 and Dsc1–3) each of which are the cad), desmocollin 3 (Dsc3), and desmoglein 3 (Dsg3), lacking a large part of their extracellular product of individual genes. Although the isoforms are distributed in a cell type and differentiation domains and containing c-myc tag at the C-terminal ends (E-cadnEC, Dsg3nEC, Dsc3nEC). specific manner, their relative functional specificities remain largely enigmatic. Previously, we and We initially failed to obtain Ad which express those mutants probably because the truncated others reported that the pemphigus foliaceus antigen, desmoglein 1 (Dsg1), can bind multiple molecules were toxic to 293 cell. Therefore, loxP flanked stuffer was interposed between promoter plakoglobin (Pg) molecules and that Pg:Dsg1 complexes exist at a 6:1 stoichiometry. This complex and mutants to prevent the expression of the products. Coinfection of Ad expressing Cre removed is unusual in that the classical cadherin, E-cadherin, as well as desmocollin (Dsc2a), were shown the stuffer and turned on the expression of the mutant cadherins. Transfection efficiency to a to associate with Pg in a 1:1 complex. In order to determine whether this 6:1 stoichiometry is cultured keratinocyte cell line, HaCaT cell, was almost 100%. After infection with Ad, adherence unique to the Dsg1 isoform, we stably coexpressed myc-tagged Pg and Dsg2 in nonadhesive L- junctions (AJ) and desmosomes (DS) were observed by dual immunofluorescence staining with cell fibroblasts. Immunoprecipitation of the cadherin/catenin complex revealed a Pg:Dsg2 ratio antib-catenin Ab as a marker of AJ, antidesmoplakin Ab as a marker of DS, and antimyc Ab. which did not exceed 1:1. This result suggests an important functional difference between the When Ad was infected to HaCaT cells cultured in high calcium condition, E-cadnEC and desmoglein cytoplasmic tails. Furthermore, a molecule chimeric for Dsc2/Dsg1, where the catenin DscnEC caused disruption of adherence junctions with morphological changes of cells, whereas binding domain of desmocollin is followed by the C-terminal unique region of Dsg1 failed to disruption by DsgnEC was limited to desmosomes. When Ad was infected to HaCaT cells reconstitute the 6:1 complex, suggesting that this domain is not sufficient to confer the ability to cultured in low calcium, induction of AJ by calcium shift was blocked by E-cadnEC and bind multiple plakoglobin molecules. This study represents the first direct evidence for isoform DscnEC, but not by DsgnEC. Induction of DS was inhibited by all three truncated constructs. specific differences among the desmosomal cadherin/catenin complexes, suggesting a means by These results indicate that the dominant negative effect of Dsg was restricted to DS, while that which the various desmogleins may make distinct contributions to the epidermal differentiation of Dsc was seen in both AJ and DS, suggesting that Dsc may interact components of both AJ and program. DS and play a role to regulate the formation of DS after establishment of AJ. 528 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

031 032 A Catenin P120 is a New Member of Desmosomal Constituents and Bound to Desmoglein 3 in Loss of Cadherin Function Differentially Affects Markers of Terminal Differentiation in Cultured Cultured Keratinocytes Human Keratinocytes Y. Aoyama, M. Sato and Y. Kitajima M. D. Hines, H. C. Jin, M. J. Wheelock,* and P. J. Jensen Department of Dermatology Gifu University School of Medicine, Gifu, Japan Department of Dermatology, University of Pennsylvania, Philadelphia, Pennsylvania; and *Depart- A catenin p120, which consists of four isoforms with molecular weights of 120 kDa (1A), 115 kDa ment of Biology, University of Toledo, Toledo, Ohio (1B), 105 kDa (2A) and 95 kDa (2B), is a cadherin-associated Src substrate and its phosphorylation Cadherin function is required for normal keratinocyte cell–cell adhesion and stratification. We of tyrosine residues is known to correlate with the reduction of adherens junctions in ras- have investigated whether cadherin activity also contributes to the regulation of normal keratinocyte transformed cells or cancer cells. This molecule, however, is not known whether it is associated differentiation, as measured by western blot analyses of differentiation-related proteins. Cultures with desmosomal cadherins. In this study, we determined whether p120 is associated with Dsg3 of neonatal foreskin keratinocytes were incubated with neutralizing monoclonal antibodies against and desmosomes in cultured human keratinocytes (NHK) and human squamous carcinoma cell line E-cadherin and/or P-cadherin under conditions that normally induce differentiation (i.e. elevated cells (DJM-1). Cultured keratinocytes were studied by immunofluorescence and immunoelectron extracellular Ca2ϩ). In control cultures (incubated with no antibody or with irrelevant cell surface microscopy using anti-p120 monoclonal antibody (pp120). Biochemically cells were solubilized antibodies), Ca2ϩ elevation induced an increase of transglutaminase 1 (TGase) and filaggrin, as with 1% Brij buffer and immunoprecipitated with anti-Dsg3 (AHP319), anti-E-cadherin (clone expected. Incubation with antibody against E-cadherin or P-cadherin did not affect this Ca2ϩ- 36), and anti-p120 (PP120) antibodies and detected by western blotting with anti-phosphotyrosine induced increase in TGase; however, incubation with both anti-E plus anti-P-cadherin antibodies antibody (25,2G4) as well as these antibodies. To determine subcellular distribution of p120, cells together led to an inhibition of TGase expression. Ca2ϩ-induced filaggrin expression was not were fractionated into cytosol (phosphate-buffered saline soluble), membrane (detergent soluble) consistently affected by antibody to P-cadherin; however, in the presence of antibody to E- and cytoskeleton (detergent insoluble) fractions. By immunofluoresenece microscopy, 0.3% Triton cadherin, there was a larger increase in filaggrin, over that induced by Ca2ϩ alone. We also X100-treated cells grown on glass coverslips showed dotted or linear distribution of p120 at cell– examined bcl-2 family members. Levels of the antiapoptotic factors bcl-2 and bag-1 were increased cell contacts. Immunoelectron microscopy revealed that p120 was localized at the cytoplasmic by Ca2ϩ elevation; however, in the presence of anti-E-cadherin or anti-P-cadherin antibodies, side of desmosomes. Biochemically, isoforms 1B and 2 A were found to be coimmunoprecipitated this increase was attenuated. In contrast, the pro-apoptotic factors bad and bak were unaffected with anti-Dsg3 antibody, suggesting that p120 was associated Dsg3 in NHK. Isoforms 1 A and by Ca2ϩ elevation in the presence or absence of either one or both anticadherin antibodies. These 2B were major p120 and also associated with Dsg3 in DJM-1. p120 was distributed in all data suggest that E-cadherin and P-cadherin contribute to the orderly progression of terminal subcellular fractions. Major tyrosine-phosphorylated isoform was 2 A. These results indicated for differentiation in the epidermis in multiple ways, which depend upon the particular differentiation the first time that p120 was a new member of desmosomal consitituents and bound to Dsg3 as marker and cadherin under study. well as E-cadherin, like plakogobin.

033 034 Ultraviolet and Cesium Irradiation Enhance Basal Cell Carcinoma Formation in Patched Heterozy- Murine Suppressor of Fused Controls Sonic Hedgehog Signaling by Modulating Activity of Gli gote Knock-out Mice Transcription Factors M. Aszterbaum, J. H. Epstein, A. E. Oro,* M. P. Scott,* and E. H. Epstein Jr Q. Ding, S. Fukami, X. Meng, M. Nakafuku, H. Sasaki, C. C. Hui and A. A. Dlugosz Department of Dermatology, University of California, San Francisco, California; *Department of Department of Dermatology and Comprehensive Cancer Center, University of Michigan, Ann Developmental Biology and Genetics and the Howard Hughes Medical Institute, Stanford Arbor, Michigan; The Hospital for Sick Children, Toronto, Canada; Department of Neuroscience, University School of Medicine, Stanford, California University of Tokyo, Tokyo, Japan; and Institute of Molecular and Cellular Biology, Osaka PATCHED (PTC) gene mutations occur both in patients with basal cell nevus syndrome (BCNS), University, Osaka, Japan an autosomal dominant syndrome characterized by a complex phenotype including multiple basal Spatially and temporally restricted Sonic hedgehog (Shh) signaling is required for hair follicle cell carcinomas (BCC), and in sporadic BCC. BCC are the most common human cancer and are morphogenesis, while constitutive activation of this pathway is associated with formation of basal induced by ultraviolet irradiation and, especially in BCNS patients, by ionizing radiation. Ptc cell carcinoma. Gli transcription factors mediate responses to Shh and are likely to play important heterozygote knock-out (KO) mice develop medulloblastomas and rhabdomyosarcomas, both of roles in normal as well as pathological processes regulated by this pathway. Although little is know which occur in higher frequency in BCNS patients, but have not yet been reported to develop of the mechanisms by which the Shh signal is transduced in vertebrate cells, a multiprotein BCC. We have identified a high incidence of BCC in mature 12 mo-old Ptc heterozygote exon complex is implicated in the transduction of the Drosophila Hedgehog signal. This complex 1 and 2 KO mice; this is the first mouse model of BCC tumorigenesis in viable adult animals. contains at least five proteins including a Gli homologue Cubitus interruptus (Ci) and Suppressor Notably, when the mice were challenged with 12 mo of UV irradiation (three times per week) of fused [Su(fu)], which interacts with Ci. Here we report the isolation of a mouse Su(fu) [mSu(fu)] we observed an average 6-fold increase in tumor incidence (12.66 vs 1.75 tumors per standard cDNA which encodes a 53-kDa PEST sequence-containing protein. Overexpression of mSu(fu) sample area) and a 100-fold increase in average tumor size. A single dose of cesium irradiation inhibited transactivation of a Gli binding-site reporter in cells cotransfected with a Shh expression (1–4 Gy) also caused a significant increase in the number of tumors per sample area (5.0 vs 1.75). vector. Several lines of evidence suggest that mSu(fu) modulates Shh signaling at the level of Gli A lacZ reporter insert in the exon 1 and 2 KO Ptc region provided a powerful in situ method for transcription factors. (i) mSu(fu) inhibited transcriptional activity of cotransfected Gli1 or Gli2. (ii) detection of Ptc promoter activation. High levels of b-galactosidase staining indicative of Ptc Using epitope-tagged proteins, mSu(fu) coimmunoprecipitated with Gli1 and Gli2. (iii) Yeast promoter activation occurred specifically in mouse BCC, hair papillae, and cutaneous nerve cells two hybrid analysis confirmed a direct interaction between mSu(fu) and Gli2. In addition, but no staining occurred in normal epidermal cells. This is consistent with the finding of increased immunofluorescence staining revealed that while Gli1 is normally present in the nucleus, PTC mRNA in human BCC. Immunohistochemical techniques confirmed that mouse BCC overexpression of mSu(fu) leads to the cytoplasmic sequestration of Gli1, which may provide the have an antigen profile identical to that of human BCC including decreased a6 and b4 integrin, mechanism by which mSu(fu) represses Gli transactivation. Taken together, these results establish BP180, and LAM5 antigens and high levels of keratin 14 antigen. Therefore we conclude that, mSu(fu) as a novel component of the Shh pathway, and strongly suggest that it functions as a as in patients with BCNS, the Ptc KO mice have a high incidence of BCC at a mature age and negative regulator of Shh signaling through cytoplasmic retention of Gli proteins. that UV and gamma irradiation result in significant enhancement of BCC carcinogenesis.

035 036 Analysis of Human Basal Cell Carcinoma Gene Expression at a Genomic Scale Via cDNA Mic- Laser-Based Microdissection and Molecular Analysis of Germinal Center-and Interfollicular roarrays Lymphocytes in Primary Cutaneous Follicular Lymphoma (Follicle Center Lymphoma, Follicular H. Fan, C. T. Barry, S. C. Baek, Q. Lin, P. O. Brown and P. A. Khavari Type) VA Palo Alto and Stanford University, Stanford, California L. Cerroni, E. Arzberger, B. Pu¨tz and H. Kerl Basal cell carcinoma (BCC) is characterized by a proliferative expansion of basal epidermal cells Department of Dermatology, University of Graz, Graz, Austria with recent progress linking it to the hedgehog signaling pathway. In spite of these advances, a It has been suggested that cases of primary cutaneous B-cell lymphoma (PCBCL) showing a broad perspective on the gene expression abnormalities characterizing BCC has been unavailable. prominent follicular pattern represent either marginal zone lymphomas with reactive germinal In an effort toward such an understanding, we sought to determine the gene expression differences centers or cutaneous pseudolymphomas. In order to establish whether a true primary cutaneous between BCC and normal skin at a genome-wide scale. mRNA was prepared from freshly excised follicular lymphoma exists, we studied lesions from 12 patients with PCBCL with follicular pattern BCC tumors and site matched normal skin control from the same patient. mRNA was then using the polymerase chain reaction (PCR) analysis of immunoglobulin heavy chain (IgH) genes reverse transcribed and labeled using fluorescent nucleotides followed by hybridization to a total after laser-based microdissection of the specimens. PCBCL was defined as presence of disease of µ12,500 arrayed genes. 149 genes whose expression was either markedly increased [113 genes] confined to the skin for at least 6 mo after complete staging procedures. Clinically patients or reduced [36 genes] in BCC versus normal skin were selected for further study, including genes presented with nodules and tumors located on the head (n ϭ 9), trunk (n ϭ 2), or both (n ϭ 1). involved in gene regulation, apoptosis, cell cycle promotion, matrix degradation as well as ESTs Histology showed dense infiltrates of lymphocytes with prominent follicular pattern throughout of unknown function. Genes whose differential expression was confirmed by northern blotting the entire dermis and subcutaneous tissues. Routinely fixed sections of tissue were stained with were then used to probe an additional panel of BCC lesional/site matched normal skin mRNA methyl-green for laser-based microdissection and PCR analysis. The laser-based system is composed specimens from six separate patients to confirm reproducibility of differential expression. Included of a pulsed UV-laser of high beam quality (nitrogen laser, wavelength 337 nm; PALM, Germany) among genes strongly upregulated in BCC are the DCC tumor suppressor gene, the EPH receptor focused through an inverse microscope into the tissue plane. In all cases, 80–100 cells each from tyrosine kinase and MMP-1, with the latter expressed 126-fold higher in BCC than in normal four to six different germinal centers and four to six different interfollicular areas were isolated skin as confirmed by northern analysis. Included among the genes strongly repressed in BCC are using the following procedure: with the UV-laser beam a circle was cut around the target cells, chitinase and c-fos, with the latter’s expression reduced by over 16-fold in BCC to nearly resulting in the complete separation of them from neighboring tissues. With the aid of a motorized undetectable levels. These data on the global patterns of gene expression in human BCC form a micromanipulator and a thin injection needle the cells were then scraped from the coverslip and basis for further insights into the pathogenesis of this tumor and for understanding basic mechanisms prepared for PCR analysis of the IgH gene using primers described previously. After gel of epithelial growth regulation. electrophoresis one or two bands of the same length were detected in samples from different germinal centers in six cases, indicating the presence of the same monoclonal population of cells. The other six cases showed a polyclonal pattern of germinal centers with one or more bands of different length (in one of these cases monoclonality of germinal centers could be demonstrated by immunohistochemical analysis of immunoglobulin light chains). Cells from interfollicular areas revealed a polyclonal pattern in all 12 cases. Our results clearly demonstrate that true primary cutaneous follicular lymphomas exist. The advantage of the laser-beam microdissection method is that contamination with DNA from neighboring cells can be avoided, thus allowing a precise correlation of PCR results with morphologic and immunohistochemical features of the lesions. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 529

037 038 Induction of Th2-Polarized Humoral Response by IL-4-Transduced Dendritic Cells Secondary Lymphoid-tissue Chemokine (SLC) and CCR7 Participate in the Emigration Pathway S. Hayashi and A. Takashima of Mature Dendritic Cells from the Skin to Regional Lymph Nodes Department of Dermatology, UT South-western Medical Center, Dallas, Texas H. Saeki, N. Yamada, M. J. Brown,* A. M. Moore and S. T. Hwang Dendritic cells (DC) play key roles in the initiation of cellular and humoral immune responses. It Dermatology Branch and *Experimental Immunology Branch, National Cancer Institute, Beth- is generally believed that DC preferentially induce ‘‘Th1-biased’’ responses, by the elaboration of esda, Maryland IL-12 and other Th1 cytokines. If this is correct, DC that are engineered to over-produce a Th2 Skin-derived dendritic cells (DC) emigrate to regional lymph nodes (LN) during immune responses cytokine should polarize the response toward Th2. To test this hypothesis, we introduced the IL- via afferent lymphatic channels. SLC, a CC chemokine, is expressed by high endothelial venules 4 cDNA into the mature DC line XS106 (derived from A/J mice). At 24 h after SuperFect- in secondary lymphoid organs and mediates chemotaxis of lymphocytes via its receptor, CCR7. mediated transfection, only small fractions (Ͻ 1%) of cells showed marked intracellular IL-4 Because mRNA for SLC is expressed by lymphatic endothelial cells, we hypothesized that it deposition, as measured by FACS analysis. On the other hand, relatively large amounts of IL-4 might be involved in the recruitment of mature skin-derived DC to dermal lymphatics. (up to 2 ng per ml per 106 cells in 14 h) were detected by ELISA in culture supernatant from Immunofluorescence microscopy was performed with a polyclonal antimurine SLC antibody. the IL-4-transfected XS106 cells (IL-4-XS). By contrast, no IL-4 was detected in culture Chemotaxis assays were performed using a modified Boyden chamber. Migratory skin-derived supernatant from nontransfected XS106 cells or the XS106 cells transfected with vector alone (v- DC were obtained from murine skin explants. CCR7 mRNA was detected by RT-PCR. Dual- XS). We next compared IL-4-XS DC vs v-XS DC for their in vivo function. In the first protocol, label fluorescence confocal microscopy using anti-SLC and MHC class II antibodies revealed A/J mice (five mice per group) received weekly s.c. injections of DC (2 ϫ105 cells per injection). MHC class II positive cells with dendritic morphology within SLC-staining lymphatic channels Mice immunized with KLH-pulsed IL-4-XS cells or KLH-pulsed v-XS cells produced significant in mouse dermis. SLC was a potent in vitro chemoattractant for cultured skin-derived DC and amounts of KLH-reactive IgG1 and IgG2a within 3 wk after the initial injection. Importantly, stimulated emigration of MHC class II positive DC from skin explants by an average of 2.5-fold. the serum level of IgG2a was significantly (p Ͻ 0.05) higher in the v-XS group. Conversely, the XS52, an immature murine dendritic cell line, showed no basal expression of CCR7 and did not serum level of KLH-reactive IgE (which became detectable 5 wk after the first injection) was respond to SLC in chemotaxis experiments. With maturation, however, CCR7 levels were up- significantly (p Ͻ 0.05) higher in the IL-4-XS106 group. No humoral response was inducible by regulated and cell migration could be stimulated up to 12-fold with SLC. In vivo, injection of nonpulsed XS106 DC or NS46 fibroblasts pulsed with KLH, documenting antigen-requirement TNF-a or IL-1b into mouse skin dramatically stimulated CCR7 mRNA expression in epidermal and DC-specificity. In the second protocol, we challenged the mice with 100 µg KLH 6 d after the LC. Function-blocking anti-SLC antibodies did not affect LC egress from the epidermis. However, initial injection; footpad swelling response was comparable between the IL-4-XS (4.6 Ϯ 0.9 ϫ10–2 anti-SLC antibodies administered systemically blocked the in vivo migration of 51Cr-labeled, skin- mm) and the V-XS groups (4.2 Ϯ 0.8). Again, no response was observed in mice immunized derived DC injected into footpads to draining LN by 50%. In conclusion, SLC is expressed in with nonpulsed XS106 DC or KLH-pulsed NS46 fibroblasts. When these mice received two dermal lymphatics and is a potent chemoattractant for mature DC. We show that proinflammatory additional DC injections after challenge, elevated production of KLH-reactive IgE was noted in cytokines up-regulate the expression of CCR7 in epidermal LC and that SLC has a minimal role the IL-4-XS group as early as 3 wk after the initial injection. Thus, Th2-polarized humoral in directing LC trafficking out of the epidermis. Our in vivo data suggest that SLC directs migration immune response is readily inducible by IL-4-transduced DC. These observations imply that of DC in dermis into lymphatics. Thus, we provide the first direct in vivo evidence that SLC and administration of antigen-pulsed, cytokine gene-transduced DC may become a novel treatment CCR7 participate in the emigration of DC from peripheral tissue to LN via lymphatics. modality to selectively switch or correct the balance of Th1/Th2 responses to a given (pathogenic) antigen in patients with inflammatory, allergic, or infectious diseases.

039 040 Molecular Cloning and Functional Characterization of a Novel Dendritic Cell-Associated Type I Prognostic Predictors of Atopic Eczema Transmembrane Protein H. C. Williams and D. P. Strachan S. Shikano, M. Bonkobara, P. Zukas and K. Ariizumi Dermato-Epidemiology Unit, Queen’s Medical Centre, Nottingham and Department of Public Department of Dermatology, University of Texas South-western Medical Center, Dallas, Texas Health Sciences, St. George’s Hospital Medical School, London, UK In searching for dendritic cell (DC)-specific genes by subtractive cDNA cloning using XS-DC Using cohort data from 6877 children in the 1958 British National Child Development Study, lines, we have identified a novel molecule, designated 2B4. The mRNA (3.4 kb) was expressed we have investigated a range of factors which might predict persistence of eczema into teenage preferentially by XS-DC lines in northern blotting. The gene encodes a putative type I years. Of 571 children with examined or reported eczema by the age of 7 y, those with eczema transmembrane glycoprotein of 574 amino acids, including a RGD tripeptide and a proline-rich at the age of 16 (examined or reported) were considered to have persistent eczema (n ϭ 150). domain in the extracellular domain. Because these components are associated with cell–cell These were compared to the remaining 421 children who did not have persistent eczema with attachment and protein–protein interactions, respectively, we hypothesized that 2B4 may serve as respect to a range of perinatal, childhood and social factors. A history of hay fever or asthma, an adhesion molecule. To test this hypothesis, a recombinant protein, 2B4-IgG, was produced onset of eczema in the first year of life and a history of childhood whooping cough were strongly consisting of the extracellular domain of 2B4 and the Fc portion of human IgG. When employed associated with persistent eczema (all p Ͻ 0.001). No significant prognostic associations were in binding studies, 2B4-IgG was found to bind to XS-DC, NS-fibroblast and Pam 212-keratinocyte shown with sex of child, birth order, birth weight, gestational age, breast feeding, smoking during lines, but not to macrophage, B-cell, or T cell lines. XS-DC strongly bound to 2B4-IgG-coated pregnancy, family size, social class, region of residence, or a history of other childhood infections. plates, but not to plates coated with an irrelevant recombinant protein, TNFR-IgG. This binding Other retrospective studies have suggested that early onset of eczema and inhalant allergy in was blocked by soluble 2B4-IgG (90% inhibition) and by RGD peptide (50%), but not by TNFR- childhood are markers for an increased chronicity of atopic eczema into adulthood. Our post hoc IgG, an irrelevant tripeptide, or EDTA. Adherence of XS-DC to NS-fibroblasts was also blocked finding of increased chronicity of eczema in children with past whooping cough requires cautious by soluble 2B4-IgG (40%). We conclude that 2B4 is a unique type I transmembrane protein interpretation and confirmation in other studies. expressed preferentially by DC. In vitro, 2B4 appears to function as an adhesion molecule through RGD-mediated and calcium-independent mechanisms. Finally, the ligand for 2B4 is expressed on DC, fibroblasts and keratinocytes. Our findings provide the basis for studying a potentially important role of 2B4 in DC migration, homing, and cell–cell interactions. In view of our new results, we propose to rename this novel molecule, DC-CAM.

041 042 p63, A p53 Gene Family Member that is Essential for Limb Development and Morphogenesis of Functional Involvement of Stat3 in Skin Remodeling Including Hair Cycle and Wound Healing the Epidermis and Hair Follicles S. Sano, S. Itami, K. Takeda,* M. Tarutani, Y. Yamaguchi, H. Miura, K. Yoshikawa, S. Akira,* A. A. Mills, B. Zheng, X. -J. Wang, H. Vogel, D. R. Roop and A. Bradley and J. Takeda† Department of Molecular and Human Genetics, Howard Hughes Medical Institute and Institute Department of Dermatology, Osaka University Medical School, Japan; *Department of Biochem- for Medical Genetics, Baylor College of Medicine, Houston, Texas; Departments of Cell Biology istry, Hyogo College of Medicine, Japan; †Department of Environmental Medicine, Osaka and Dermatology, Baylor College of Medicine, Houston, Texas University Medical School, Japan We have recently identified a mouse gene, p63, which shares considerable homology with the Many growth factors and their receptors play key roles in skin and hair morphogenesis, and DNA binding domain of the p53 tumor suppressor gene. In order to functionally assess the remodeling such as wound healing and hair cycle. It remains, however, still unclear as to what requirement for p63 during embryonic development, we have disrupted the endogenous p63 intracellular signaling molecules are required for specific cellular events. To elucidate the biological locus using homologous recombination. p63-deficient mice are viable at birth and exhibit striking roles of Stat3 in the skin, conditional gene targeting was performed since conventional knockout craniofacial, limb, and skin defects. The hindlimbs are completely absent, while forelimbs are of the gene leads to embryonic lethality. Keratinocyte-specific Stat3 deficient mice were generated truncated at their distal elements. Limb buds of p63-null embryos misexpress Lmx-1, lack a by crossing mice with Cre transgene driven by keratin 5 promoter (K5-Cre) and mice with floxed defined apical ectodermal ridge (AER), and fail to express Fgf-8. The skin of p63-homozygous Stat3 (Stat3flox). K5-Cre: Stat3flox/– mice, whose epidermal and follicular keratinocytes lacked mutant mice is completely devoid of hair follicles. The epidermis of p63-deficient newborn mice functional Stat3, were viable and their development of epidermis and hair follicles appeared lacks stratification, does not express differentiation markers, and exhibits a marked increase in normal. Surprisingly, the second hair cycle was interrupted at anagen stage, and wound healing water loss as compared to the epidermis of wild-type siblings. Since the epidermis of the was severely retarded. Growth factor-dependent in vitro migration of Stat3-disrupted keratinocytes homozygous mutants consists of only a single cell layer, epidermal development must have arrested was severely impaired, while the proliferative response was not affected. These results strongly before E15.5 when the epidermis initially stratifies. In normal epidermis, keratin 14 (K14) suggest that Stat3 plays a critical role in mediating a signal for migration but not proliferation of expression is first detected at E9.5, and hair follicles begin to develop around E14.5. However, keratinocytes, and is essential for skin remodeling including wound healing and hair cycle. the epidermis of p63 homozygous mutant animals exhibits only faint and patchy staining of K14, and does not form placodes, the precursors of hair follicles. Thus, epidermal development apparently ceases around E9.5 in p63-deficient mice, consistent with the onset of p63 expression in the ectoderm of wild-type mice at E9.5. This study indicates that, in contrast to p53, p63 is essential for limb development and morphogenesis of the epidermis and hair follicles. 530 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

043 044 Development of a Transgenic Mouse Model That Allows Focal Deletion of Genes in the Epidermis HIV-1 Primary Isolate Infection of Immature Langerhans Cells within Epithelial Sheets: A Novel T. R. Berton, X. J. Wang, G. Shutz, S. Tsai and D. R. Roop Model to Study Primary HIV Infection Departments of Cell Biology and Dermatology, Baylor College of Medicine, Houston, Texas; and S. S. Cohen, M. Qalbani, E. A. Aquilino, A. R. Neurath and A. Blauvelt the German Cancer Research Center, Heidelberg, Germany Dermatology Branch, NCI, Bethesda, Maryland; New York Blood Center, New York, New York Conventional transgenic approaches to determine the effects of loss-of-function mutations via Although unprotected sex is the most common mode of HIV transmission worldwide, the biologic germ line deletion, often result in embryonic lethality and prevent an assessment of the effects on events which underlie this process are poorly understood. In this study, we have developed a epidermal development, which occurs late in gestation. In addition, germ line deletions of tumor novel model for primary HIV infection and have used this model to evaluate potential topical suppressor genes, such as p53, have hindered attempts to assess malignant progression of skin antimicrobicides that could block sexual transmission of HIV. Suction blister roofs (i.e. epidermal tumors, since p53 null mice are predisposed to develop spontaneous internal malignances and sheets) containing Langerhans cells, purported initial targets for HIV in genital mucosa, were often die from these lesions before skin tumors develop. We have developed a transgenic mouse obtained from normal-appearing skin of healthy volunteers and exposed for 2 h to various dilutions model that allows focal deletion of a target gene in the epidermis after topical application of the of a panel of early passage-primary and laboratory-adapted HIV-1 isolates. Sheets were then progesterone antagonist, ZK 98.734. To achieve epidermal specific knockouts, two transgenic washed to remove excess virus and floated in culture media for 2 d. HIV-infected Langerhans mouse lines were required. One transgenic mouse line expresses Cre recombinase in the epidermis. cells emigrated from sheets and transmitted high levels of infection to cocultured allogeneic By fusing the Cre recombinase with a truncated progesterone receptor the recombinase activity CD4ϩ T cells (maximum viral loads of 50–500 ng of HIV p24 per ml of supernatant by ELISA). is sequestered in the cytoplasm until ZK induces translocation to the nucleus. The other transgenic We then tested 3-hydroxyphthaloyl-b-lactoglobulin (3-HP-b-LG), B195, and AOP-RANTES mouse line was engineered to contain the CAT-Z reporter gene, which directs expression of the (three new potential topical antiviral agents) for their ability to block Langerhans cells infection lacZ gene upon Cre-mediated excision of the upstream CAT gene that is flanked by loxP sites. within roofs. Interestingly, preincubation of roofs with each drug for 20 min blocked subsequent Newborn double transgenic mice were topically treated with either ZK (10 µg) or ethanol. HIV infection of Langerhans cells in a dose-dependent manner (85%–100% inhibition at higher Twenty hours after a single treatment of ZK we observed b-galactosidase activity in the epidermis, doses compared to vehicle control or no drug conditions). In addition, preincubation of drugs and after seven treatments (3.5 d) b-galactosidase positive cells began to cluster. These data with immature blood-derived dendritic cells propagated in the presence of GM-CSF and IL-4 demonstrate the feasibility the focal deletion of a target gene in the epidermis of transgenic mice, (which resemble Langerhans cells in situ) inhibited HIV infection in these cells. In summary, we which will more closely mimic the sporadic occurrence of somatic mutations in human cancer. have established a novel physiologically relevant system to study primary HIV infection and have initially used it for preclinical testing of potential topical antiviral agents. Our model could prove invaluable in further understanding sexual transmission of HIV and in the development of agents designed to interfere with this process.

045 046 Activation of HIV by UVB Radiation is Inhibited by a DNA Decoy Directed Against NF-kB Direct and Indirect Roles for Transcription Factors C/EBP and AP-2 in the Regulation of Keratin P. Cruz, Jr, K. Ariizumi, I. Dougherty and B. Dawson k10 Gene Expression in Mammalian Epidermis Departments of Dermatology and Pathology, UT South-western Medical Center, Dallas, Texas E. V. Maytin, J. C. Lin, R. Krishnamurthy, N. Batchvarova, D. Ron, P. J. Mitchell and J. F. Habener UVB radiation has been shown to activate HIV in cultured cells and transgenic animals. To verify Department of Dermatology, Laboratory of Molecular Endocrinology, and Howard Hughes a similar effect in humans, we developed a quantitative PCR-based assay for measuring HIV-1 Medical Institute, Massachusetts General Hospital, Harvard Medical School, Boston; Skirball (gag) in skin. Using this assay, we have shown that UVB treatment in suberythmogenic doses Institute of Biomolecular Medicine, NYU Medical Center, New York, New York; Institute of leads to activation of HIV, as detected in lesional psoriatic skin of HIV(ϩ) patients. NF-kB is a Pharmacology, University of Zu¨rich, Zu¨rich, Switzerland key initiator of inflammation, including the cascade induced by UVB radiation. NF-kB has also The epidermis forms a vital barrier composed of stratified keratinocytes and their differentiated been implicated as an activator of viral transcription. To test the hypothesis that NF-kB plays a products. Keratin K10, an intermediate filament expressed mainly in suprabasal keratinocytes, is critical role in UVB-induced HIV activation, we examined the effect of a DNA decoy on our critical to epidermal integrity. In humans, dominant mutations in k10 cause epidermolytic experimental protocol. A 20 mer, double-stranded oligonucleotide decoy containing the NF-kB hyperkeratosis, a skin disorder typified by abnormal blistering. Here, regulation of the k10 gene binding site sequence was prepared and purified by HPLC. An irrelevant decoy of identical size promoter was examined in murine keratinocytes in vitro and in vivo. In cultured keratinocytes, and G:C content, but with shuffled sequence, was used as control. Lesional skin specimens (3 mm) transcription factors C/EBPa and C/EBPb each activate the k10 promoter via multiple binding from HIV(ϩ) patients with psoriasis were procured and incubated (3 h) with decoy (1 or 5 µM), sites, as shown by site-directed mutagenesis and transactivation analyses. C/EBPb and transcription irrelevant decoy, or media alone, prior to exposure to UVB (200 J per m2) or to sham irradiation. factor AP-2 are abundant in basal keratinocytes in vivo, whereas AP-2 is downregulated in Specimens were then reincubated (16 h) with decoy, irrelevant decoy, or media alone, as designated suprabasal layers where C/EBPa is abundant. In c/ebpb nullizygous mice, k10 expression is previously. RNA was extracted and HIV measured. Consistent with previous studies, UVB- upregulated in basal keratinocytes coincident with loss of AP-2 and upregulation of C/EBPa. exposed skin (81 copies per µg RNA) showed a 6- to 10-fold rise in HIV count compared to Analysis of the ap-2 gene promoter revealed C/EBP-responsive elements that may account for unirradiated skin. Irrelevant decoy-treated, UVB-exposed skin (79 copies per µg RNA) had a the loss of expression in c/ebpb null skin. In the epidermis of ap-2 null mice, C/EBPb expression similarly high viral count. By contrast, decoy-treated, UVB-exposed skin (11 copies per µg RNA) is normal whereas C/EBPa and k10 expression are upregulated. Together, these data suggest a exhibited a 90% reduction in HIV load. We conclude that UVB-induced activation of HIV is regulatory model in which C/EBPb activates and maintains AP-2 expression in undifferentiated mediated by NF-kB, and that such activation can be abrogated by a DNA decoy containing the keratinocytes, whereas AP-2 represses C/EBPa in these cells. In response to differentiation signals, binding site sequence of the transcription factor. These findings have enormous implications for loss of AP-2 expression leads to derepression of the c/ebpa promoter and activation of k10 the management of HIV-infected patients. gene expression.

047 048 NF-kB-Directed Transcription is Normally Localized to Non-Proliferating Cells Within Epidermis A Novel Sorting Method Yields a Pure Population of Viable Stem Cells which Reforms a and is Strongly Induced by Ultraviolet Injury But Not by Wounding Complete Epidermis with Long-term Recombinant Gene Expression A. M. Gervin, S. Memet,* R. A. Freiberg, A. Israel,* and P. A. Khavari J. R. Bickenbach VA Palo Alto and Stanford University, Stanford, California; and *Pasteur Institute, Paris, France Department of Anatomy and Cell Biology, The University of Iowa, Iowa City, Iowa NF-kB gene regulatory proteins have recently been appreciated as necessary for normal negative Continuously renewing epithelia are maintained by small undifferentiated stem cells. It is believed growth regulation in epidermis and NF-kB DNA binding activity is strongly induced by ultraviolet that they are the only cells that continuously replicate their DNA and remain in the tissue for (UV) radiation, however, the localization of NF-kB activity in skin in either setting has not yet life. Thus, if gene therapy regimes for genetically inherited skin diseases are to be effective, stem been defined. To localize the cellular site of action of this transcription factor within living skin, cells must be targeted. Although it has been known for decades that epidermal stem cells can be we utilized three independent lines of transgenic mice containing NF-kB binding sites driving identified by long-term retention of a nuclear label, isolating a pure population of stem cells has expression of the E. coli lacZ gene. X-gal staining of normal skin of these mice localized NF-kB- been problematic, until now. Using a modified Hoescht dye, a modified sheath fluid, and directed gene induction primarily within cells of the suprabasal epidermis. Consistent with this specifically defined gating on a Coulter EPICS 753 with two argon lasers, we sorted dissociated finding, double staining experiments with the Ki-67 proliferation marker indicated that NF-kB epidermal cells into three viable subpopulations: (i) a 96% pure population of small undifferentiated activity occurs almost exclusively within nonproliferative cells. While prior studies have demon- label-retaining stem cells (ii) small transit amplifying basal cells that are not label-retaining, and strated the induction of NF-kB DNA binding activity in skin tissue extracts after UV radiation, (iii) large K5/K14 positive basal cells. Fraction 1 (stem cells) represented 3%–4% of the basal cell the cellular sites of NF-kB action in this process are unknown. To determine the localization of population; fraction 2 (transit amplifying cells) nearly 87%; and fraction 3 µ10%. Both the stem NF-kB-directed gene expression following ultraviolet injury, we irradiated transgenic mice with cell and the transit amplifying cell fractions retained proliferative capacity and individual cells from 50 mJ per cm2 UVB and examined skin for target gene expression at 12, 24, and 48 h. NF-kB- these fractions formed large colonies in culture. Further, both populations could be expanded in directed gene expression is strongly induced in basal and suprabasal cells as early as 12 h after UV. culture. Cells from fraction 3 did not grow, indicating that although these basal cells still expressed This finding is consistent with the growth arrest observed in basal cells following UV injury; K5 and K14, they had already left the proliferative pool and begun to differentiate. Cells from given NF-kB’s known role in epithelial growth inhibition, it also implicates NF-kB as a potential both the stem and transit amplifying cell fractions could be transduced with a retroviral vector mediator of this process. Interestingly, NF-kB-directed transcription was not detected in parallel and used to reform an epidermis that expressed the appropriate differentiation markers. However, experiments using traumatic wounding as a potential trigger, arguing for the specificity of physical only the epidermis from the stem cell fraction continued to grow and express the reporter alkaline stimuli sufficient to induce NF-kB-directed gene expression in skin. These data indicate that NF- phosphatase gene for over 6 mo in organotypic culture. The epidermis from the transit amplifying kB is biologically active in nonproliferative cells of the epidermis and that its transcriptional activity cell fraction completely differentiated by 2 mo. Thus, this novel sorting method yields a viable is rapidly induced in the proliferative basal compartment following UV injury. pure population of stem cells that can now be tested in gene therapy regimes and used to determine unique markers for epidermal stem cells. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 531

049 050 Properties of Immortal Cell Lines Arising from Human Epidermal Keratinocytes and Mesothelial The Shedded Ectodomain of Collagen XVII/BP810 is Targeted by Auto-antibodies in Blistering Cells Stably Transfected to Express hTERT (Human Telomerase Catalytic Subunit) Skin Diseases J. G. Rheinwald, M. A. Dickson, W. C. Hahn, R. A. Weinberg, V. Ronfard, F. P. Li and J. Y. Wu H. Schumann, J. Baetge, K. Tasanen, F. Wojnarowska, H. Scha¨cke, D. Zillikens and L. Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Bruckner-Tuderman Massachusetts; Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Department of Dermatology, University of Mu¨nster and Wu¨rzburg, Germany; Department of Cambridge, Massachusetts; Organogenesis, Inc., Canton, Massachusetts Dermatology, University of Oulu, Finland; Department of Dermatology, Oxford University, We have sought to determine whether expression of hTERT in two epithelial cell types, the Oxford, UK keratinocyte and the mesothelial cell, confers replicative immortality without loss of normal Collagen XVII, also known as the 180 kDa bullous pemphigoid antigen-2 (BP180), is an epidermal growth regulation and differentiation. Primary human keratinocytes from the epidermis of a adhesion protein. It exists in two forms, as the full-length transmembrane protein and as a 120- newborn (N) and from an adult (LiF-Ep), N cells previously transduced to express HPV16 E7 kDa collagenous ectodomain which is shedded from the keratinocyte surface by furin mediated (N/E7), keratinocytes from adult oral mucosa (OKF6), and mesothelial cells from adult peritoneum proteolytic processing. Here the shedded ectodomain of human collagen XVII was isolated from of (LP9) were retrovirally transduced to express hTERT. The transductants had telomerase activity keratinocyte culture medium and amnion fluid and used as an antigen in immunoassays. IgG and (TRAP assay) and their average telomere length was extended compared to untransfected and IgA autoantibodies in sera or blister fluid of patients with subepidermal blistering skin diseases empty vector controls. At the time control cells passaged in parallel became senescent, the hTERT- recognized the ectodomain. The antibody reactivity was not dependent on the native conformation transduced N, LiF-Ep, and OKF6 cells entered a 4–6 wk plateau phase of very slow division. or N-glycosylation of the ectodomain, but was abolished by collagenase-treatment of the antigen. More rapidly proliferating cells then appeared, repopulating these cultures and dividing for Ͼ50 The autoanti-bodies were affinity-absorbed from the patient sera with the ectodomain and extra doublings so far. In contrast, all cells in the LP9/hTERT and N/E7/hTERT populations incubated with NaCl-split normal skin where they bound to the blister roof, at the proximal were apparently immortal, continuously dividing for Ͼ 50 extra doublings so far. The immortal surface of the epidermis. In contrast, no reactivity was seen with collagen XVII-deficient skin or hTERT-transductants exhibit many normal cell type-specific features of growth regulation and keratinocytes. The full-length transmembrane collagen XVII and procaryotic and eu-caryotic differentiation, including EGF-dependence, TGFb-and PMA-inhibition, involucrin and K1/K10 recombinant fragments spanning the ectodomain were parallelly employed to the characterize the expression, and formation of an organized, differentiated stratified squamous epithelium in IgG and IgA reactivities in sera of 108 patients with bullous diseases. In disorders of the pemphigoid organotypic culture for the keratinocytes, EGF-dependent growth and keratin regulation for the group, 87% of the IgG autoantibodies reactive with the full-length collagen XVII also recognized mesothelial cells, and p53 increase after DNA damage for both cell types. Absent from N/hTERT the 120 kDa ectodomain. In linear IgA dermatosis, 55% of the sera and in chronic bullous cultures, however, were any large p16ink4a ϩ cells, present in normal keratinocyte cultures. These dermatosis of childhood, all sera contained IgA antibodies reactive with the soluble ectodomain. results imply that a second genetic event to inactivate the p16/cyclin-cdk/pRB, but not the p53, Use of recombinant collagen XVII fragments as antigens demonstrated that the epitopes were pathway must complement hTERT to immortalize keratinocytes, whereas hTERT alone can spread over several noncollagenous and collagenous subdomains. These data identify the shedded immortalize mesothelial cells. form of collagen XVII as an epidermal autoantigen which is a target for both IgG and IgA antibodies in subepidermal blistering skin diseases.

051 052 Desmoglein (Dsg) Compensation Hypothesis Explains Telogen Hair Loss in Dsg3 Mutant Mice A Novel Active Disease Mouse Model for Pemphigus Vulgaris: Transfer of Splenocytes from and Lack of Pathology in Neonatal Pemphigus Foliaceus Desmoglein 3 (Dsg3)-immunized Dsg3 Knockout Mice to Immunodeficient Mice Expressing Dsg3 H. Wu, A. Yan, S. Lyle, M. Mahoney, M. Shapiro, J. Wahl, M. Wheelock, G. Cotsarelis and J. M. Amagai, K. Tsunoda, K. Nishifuji, H. Suzuki,* S. Koyasu,* and T. Nishikawa R. Stanley Departments of Dermatology and *Immunology, Keio University School of Medicine, Tokyo, Japan University of Pennsylvania, Philadelphia, Pennsylvania; and University of Toledo, Toledo, Ohio In pemphigus vulgaris (PV) anti-Dsg3 autoantibodies disrupt cell–cell adhesion of keratinocytes, The desmoglein (Dsg) compensation hypothesis states that coexpression of Dsg 1 and 3 protects causing blisters. The purpose of this study was to create an active mouse model of PV and compare against pathology due to dysfunction of either one alone. This model predicts that loss of function, the phenotype to Dsg3–/– mice. Dsg3–/– mice (n ϭ 4) and Dsg3ϩ/– mice (n ϭ 3) were caused by genetic mutation or pemphigus autoantibodies, of Dsg 1 or 3 in areas where they are immunized with the extracellular domain of mouse Dsg3 expressed by baculovirus. Only Dsg3–/– not coexpressed will result in acantholysis. Dsg3–/– mice have no spontaneous skin lesions, mice produced anti-Dsg3 antibodies, suggesting that, unlike mice that express Dsg3, Dsg3–/– however, do develop loss of telogen hair due to acantholysis in the club. To examine Dsg mice do not have tolerance against Dsg3. We then transferred splenocytes of immunized Dsg3–/– expression in mouse epidermis and telogen hair we raised rabbit antibodies against mouse Dsg1 mice to RAG2–/– immunodeficient mice which express Dsg3. Circulating antibodies against and Dsg3. Specificity was confirmed by immunoblotting. Immunoflourescence staining showed Dsg3 was detected in these RAG2–/– mice, but not controls to whom splenocytes of immunized expression of Dsg1 throughout the epidermis of normal and Dsg3–/– mice, thus maintaining cell Dsg3ϩ/– mice were transferred, as early as at day 3 then increased, reaching a plateau level at adhesion even in the absence of Dsg3. However, the telogen club demonstrated only Dsg3 week 3 and persisting for over 2 mo. Direct immunofluorescence showed mouse IgG deposition without Dsg1 in normal mice and neither Dsg in mutant mice, consistent with pathology resulting on keratinocyte cell surfaces in oral and esophageal mucous membranes, epidermis and hair from loss of function of Dsg3 without compensating Dsg1. To determine why anti-Dsg1 IgG in follicles. These RAG–/– started to lose weight and show patchy hair loss at week 3–4 (n ϭ 6), PF causes blisters in adult but not neonatal skin, we examined the distribution of human Dsg 1 and histology showed oropharyngeal erosions with suprabasilar acantholysis as well as acantholysis and 3 with monoclonal antibodies raised against each. Adult epidermis showed Dsg1 throughout, of the cells surrounding the telogen club and the basal layer of the outer root sheath epithelium. but Dsg3 only in the basal cell layer. In sharp contrast, neonatal skin showed both Dsgs throughout This phenotype is identical to that of Dsg3–/– mice, suggesting that the anti-Dsg3 antibodies the epidermis, as is found in normal adult mucous membranes (which are also unaffected in produced in RAG2–/– mice cause dysfunction of Dsg3. These data demonstrate that the pemphigus foliaceus). These data suggest that Dsg3 in the superficial neonatal epidermis protects extracellular domain of Dsg3 can produce pathogenic antibodies in mice that should prove useful against pemphigus foliaceus pathology. Thus, the Dsg compensation hypothesis provides a basis for defining functional epitopes in Dsg3, and provide a model to study cellular mechanisms of for understanding the pathophysiology of pemphigus and Dsg3 mutant mice and underscores the autoantibody production in PV as well as to develop disease-specific therapeutic strategies. importance of Dsgs in maintaining cell adhesion.

053 054 Characterization by Immunoprecipitation of Non-Desmoglein 1 Pemphigus Vulgaris Antibodies Development of an Animal Model of Bullous Pemphigoid Characterized by Basement Membrane Causing Blisters in Desmoglein 3-deficient Mice Zone Separation and an Eosinophilic Inflammatory Infiltrate V. T. Nguyen, A. Ndoye, L. D. Shultz,* and S. A. Grando C. A. Egan, T. B. Taylor, M. W. Foutz, S. R. Florell, L. J. Meyer, M. J. Petersen and J. J. Zone Department of Dermatology, University of California, Davis, Californis; and *The Jackson Department of Dermatology, University of Utah, Salt Lake City, Utah Laboratory, Bar Harbor, Maine An animal model of bullous pemphigoid (BP) exists which utilizes polyclonal rabbit antibodies to The pathophysiology of pemphigus vulgaris (PV) is not limited to desmoglein (Dsg) 3 antibodies murine BPAg2. In this model, antibody administration induces a neutrophilic infiltrate and rapid because PV IgGs bind to epidermis and induce acantholysis and gross blisters in Dsg 3-knockout sloughing of the epidermis of neonatal BALB/C mice. This mouse model of BP differs from mice. In addition to the 130 kDa Dsg 3, PV patients’ IgGs uniquely recognize other keratinocyte human disease in which an urticarial phase frequently precedes blister formation and an eosinophilic proteins upon immunoblotting. Since the conformational epitope of true PV antigen(s) may be inflammatory infiltrate is seen. We have previously described 2 monoclonal antibodies (MAb 97- destroyed in the course of immunoblotting, the current study characterizes disease-causing PV 1, MAb 97-2) reactive with LABD97, which represents a portion of the extracellular domain of antibodies in the immunoprecipitation assays that employed naturally folded keratinocyte proteins. BPAg2. We passively transferred these antibodies to athymic mice grafted with human foreskin. To optimize the sensitivity and specificity of the assay, as a source of PV antigens we used: (i) Both MAb 97-1 and MAb 97-2 (n ϭ 15) or either antibody alone (n ϭ 5) were injected 125I-labeled proteins solubilized with a nonionic detergent from normal adult human epidermis; subcutaneously into grafted mice. Controls were injected with a mouse monoclonal antibody (ii) whole-cell protein extract of cultured human keratinocytes metabolically labeled with 35S- directed against a human prostate antigen (n ϭ 5). Circulating basement membrane zone (BMZ) methionine; and (iii) 125I-labeled 45 kDa ‘‘tryptic fragment’’ of Dsg 1 purified by Con A affinity antibodies were detected in all mice receiving MAb 97-1 and/or MAb 97-2, with titers A 1:80. chromatography. In the assays used, Dsg 1 maintained intact its conformational epitope, because BMZ IgG deposition was noted in the grafts of all animals except controls. BMZ deposition of it was recognized by control PV, and pemphigus foliaceus IgGs. The PV IgGs that did not C3 was variable and most consistently seen with administration of both antibodies (14 of 15 mice). crossreact with Dsg 1 were injected i.p. at 20 mg/g body weight into two different strains of BMZ separation was seen in 12 of 15 grafts from mice receiving both antibodies, two of five mice that lack Dsg 3: Dsg3null mice homozygous for a targeted mutation of the Dsg3 gene and receiving MAb 97-1 and three of five receiving MAb 97-2. BMZ separation was noted in one ‘‘balding’’ Dsg3bal/Dsg3bal mice homozygous for a spontaneous null mutation in the Dsg3 gene. control mouse in the absence of a cellular infiltrate. Eosinophils were noted in six of 15 grafts in These PV, but not normal, IgGs caused gross skin blisters with suprabasal acantholysis and stained mice receiving both antibodies and in one graft from a mouse given MAb 97-1. Neutrophils perilesional epidermis of injected Dsg 3-deficient mice in a fishnet-like, intercellular pattern. By were rare in the inflammatory infiltrate. In conclusion, monoclonal antibodies to BPAg2 are immunoprecipitation, in addition to the 130 kDa Dsg 3 target that was missing in these mice, the pathogenic to human skin grafted onto athymic mice. BMZ separation and an eosinophilic non-Dsg 1 pathogenic PV IgGs reacted with keratinocyte proteins of 45, 70, 80, 105, and infiltrate was seen most often with administration of both antibodies. This model has considerable 130 kDa. In conclusion: (i) Dsg3bal/Dsg3bal mice, as well as Dsg3null mice, can be used to study potential in the study of the pathogenesis of BP, as it closely mirrors human disease. non-Dsg 3 PV autoimmunity; and (ii) Dsg 1 antibody is not required to induce blisters in Dsg 3- deficient mice. The symptoms of PV can therefore be induced by non-Dsg 1/Dsg 3 antibodies. 532 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

055 056 Plasminogen Activator is not Required for Blister Formation in Pemphigus Requirement of CD4ϩ T Cell Collaboration for the Autoantibody Response to Desmoglein 3 Z. Wang, M. G. Mahoney and J. R. Stanley (Dsg3) in Pemphigus Vulgaris Department of Dermatology, University of Pennsylvania, Philadelphia, Pennsylvania K. Nishifuji,*† M. Kuwana,‡ T. Iwasaki,† T. Nishikawa,* and M. Amagai* Previous studies have suggested that urokinase plasminogen activator (u-PA) is required for blister Department of *Dermatology and ‡Institute for Advanced Medicine, Keio University School of formation in pemphigus vulgaris (PV) and foliaceus (PF). However, other studies have shown that Medicine, Tokyo, and †Veterinary Medical Teaching Hospital, Gifu University, Gifu, Japan down regulation of PA does not inhibit blisters induced by pemphigus IgG. To address this issue, Patients with pemphigus vulgaris (PV) have circulating IgG autoantibodies against desmoglein 3 we passively transferred PF IgG to transgenic neonatal mice that over express PA inhibitor 1 (PAI- (Dsg3), which inhibit cell–cell adhesion of keratinocytes, causing blisters. Previously we have 1), a potent inhibitor of both u-PA and t-PA, in epidermis. Even though by ELISA these mice developed an enzyme-linked immunospot (ELISPOT) assay to detect and numerate Dsg3-specific showed a 70- ϫincrease in PAI-1 in epidermis compared to nontransgenics, they developed gross autoimmune B cells. The purpose of this study was to examine roles of T cells in autoantibody and microscopic superficial epidermal blisters. To eliminate the possibility that small amounts of production in PV. We first developed in vitro autoantibody production system using peripheral residual PA activity might be adequate for blister formation, we passively transferred PV and PF blood mononuclear cells (PBMC) from patients with PV. We stimulated PBMC in an antigen- IgG to u-PA and tissue type PA (t-PA) knockout mice and litter mates. PF IgG at high (average specific way with a low concentration of PWM (0.125–0.25 µg per ml) in addition to rDsg3 4.4 mg per g) and low (average 1.1 mg per g) doses and PV IgG at high (avgerage 8 mg per g) (0.25–4 µg per ml), incubated for 7 d and detected and numerated anti-Dsg3 autoantibody- and low (avgerage 3 mg per g) doses caused gross and microscopic blisters to the same degree in producing B cells with ELISPOT assay. CD4ϩ T cell-depleted PBMC were prepared from three control (PF, N ϭ 11; PV, N ϭ 18), u-PA (PF, N ϭ 16; PV, N ϭ 16) and t-PA (PF, N ϭ 9; PV, PV patients and the number of the spots were compared with nondepleted PBMC. Anti-Dsg3 N ϭ 18) knockout mice. In all mice, PF and PV IgG caused acantholysis in the superficial and antibody production was completely lost or largely diminished when CD4ϩ T cells were depleted suprabasal epidermis, respectively, as expected. To rule out that one PA might compensate for the in the all three PV patients examined. Furthermore, we examined the effect of anti-MHC class other in the knockout mice, we bred u-PA, t-PA double knockouts. After passive transfer of PF II monoclonal antibodies on the in vitro anti-Dsg3 antibody production. In two patients, both of anti- and PV IgG these mice (PF, N ϭ 3; PV, N ϭ 3) blistered to the same degree as the single HLA-DR and anti-HLA-DQ monoclonal antibodies, in one patient, anti-HLA-DQ monoclonal knockout and control mice, and histology indicated blisters at the expected level of the epidermis. antibody, markedly suppressed the number of the spots whereas anti-HLA-DP and isotype controls These data definitively demonstrate that PA is not necessary for pemphigus IgG to induce had no apparent effect. These findings indicate that the in vitro anti-Dsg3 autoantibody production acantholysis in the neonatal mouse model of pemphigus. is mediated by collaboration between CD4ϩ T cells and B cells via an interaction of HLA-DR and/or HLA-DQ molecules and T cell receptors. This is the first demonstration of an essential role of T cells in autoantibody production in pemphigus.

057 058 The Antigenicity of the Pemphigus Foliaceus Autoantigen (PF-AG) is Differentiation-Related Transmission of Experimental Bullous Pemphigoid/Herpes Gestationis from Mother to Neonate and Depends on Carbohydrate Moities Z. Liu, X. Zhou, R. Chen and L. A. Diaz S. Ortiz-Urda, U. Mann, E. Kriehuber, K. Wolff and K. Rappersberger Department of Dermatology, Medical College of Wisconsin, and VA Medical Center, Mil- Department of Dermatology, University of Vienna, Wa¨hringer Gu¨rtel, Vienna, Austria waukee, Wiscosin Patients with pemphigus foliaceus (PF) develop autoantibodies against the extracellular domain of Bullous pemphigoid (BP) is an autoimmune disease characterized by subepidermal blistering DgI, a constitutive desmosomal cadherin type adhesion molecule; however, autoantibody-binding associated with the deposition of complement-fixing IgG at the basement membrane zone and is restricted to desmosomes present in differentiated squamous epithelia. This restrictive expression inflammatory infiltration of the lesional site. Using a passive transfer mouse model, our research pattern and the observation that DgI displays several potential N-glycosilation sites, two of which group has obtained strong evidence that the pathogenic antibodies in BP are directed against the are in close proximity to its RAL-homotypic binding domain, suggest that post-translational ectodomain of BP180, a constituent of the hemidesmosome. Herpes gestationis (HG), a disease glycosilation of DgI could account for this peculiar expression pattern of the PF-AG. In this study that occurs during the second or third trimester of pregnancy, has clinical and immunohistological we processed biochemically characterized PF-sera on normal human skin and found that binding features very similar to those of BP, and anti-BP180 autoantibodies have been linked to the was completely inhibited by blocking of sugar residues with several lectins, whereas binding with pathogenesis of this disease as well. Some neonates of HG mothers develop a transient papulovesicu- a monoclonal antibody (MoAb-Dg3.10) that detects an intracellular epitope of DgI was not lar eruption that appears to be the result of a transplacental passage of pathogenic IgG. In this afflicted. In addition, deglycosilation of cryosections of normal human skin with N-glycosidases study we addressed two questions: (i) can pathogenic anti-BP180 antibodies be transferred from also completely inhibited autoantibody binding, but did not interfere with the monoclonal mother to neonate and cause blisters? (ii) Is the transfer of disease by pathogenic anti-BP180 IgG antibody binding. These findings were supported by biochemical experiments that showed that modulated by the neonatal Fc receptor (FcRn)? Pregnant BALB/C mice were injected intradermally after deglycosilation of DgI, no staining was achieved with patients sera, but the monoclonal (i.d.) with pathogenic anti-BP180 IgG at 18, 19, and 20 d gestation (gestation in mice is 21 d). antibody detected a protein that now had a molecular weight of 145 kDa. In additional experiments The newborns from these experimental animals developed subepidermal blisters. Anti-BP180 IgG we found DgI expression in primary keratinocytes, however, PF autoantigen expression could was detected in the sera and found to be deposited at the dermal–epidermal junction in these only be observed after 24 h of organotypic growth. This peculiar expression was accompagnied neonates. In contrast, neonates from FcRn-deficient mice receiving up to four times more by the change of basal cell keratins 5/14 to keratins 1/10, indicating differentiation dependence pathogenic anti-BP180 IgG showed no signs of skin lesions and had no detectable levels of anti- of PF-AG expression as a consequence of post-translational glycosilation. BP180 IgG in circulation or bound to the dermal–epidermal junction. These results suggest that subepidermal blistering in neonates of HG patients is induced by anti-BP180 IgG and is modulated by the placental FcRn which determines the amounts of IgG reaching the baby.

059 060 Paraneoplastic Pemphigus Autoimmunity Includes Antibodies Against HD1/Plectin Anti-Type IV Collagen Antibodies in Serum from a Patient with Sub-Epidermal Blistering are C. Proby, Y. Fujii, K. Owaribe, T. Nishikawa and M. Amagai Directed Against the a5 (IV) Chain Department of Dermatology, Keio University School of Medicine, Tokyo, Japan; Department of R. F. Ghohestani, J. F. Nicolas, A. Claudy and J. Uitto Molecular Biology, School of Science, Nagoya University, Nagoya, Japan Immunodermatology Unit, Department of Dermatology and Cutaneous Biology, Jefferson Medical The characteristic antigen complex which defines paraneoplastic pemphigus (PNP) includes College, Philadelphia, Pennsylvania; INSERM U999 and Department of Dermatology, Claude desmoplakins I and II (DPI and DPII), bullous pemphigoid antigen I (BPAGI), envoplakin and Bernard University, Lyon, France periplakin – five proteins with related sequences which are all members of the plakin gene family. Collagen type IV is a heterotrimeric complex of proteins composed of a1-a6 subunit polypeptides. The purpose of this study was to determine whether HD1/plectin is also a target autoantigen in We have recently identified a novel subepidermal blistering skin disease with auto-antibodies PNP. This 500 kDa plakin protein may have not been detected as an autoantigen before directed against the type IV collagen. In the present study we have identified the specific chain because of practical difficulties associated with immunoprecipitating and immunoblotting such a of type IV collagen that harbors the auto-reactive epitopes. By immunofluorescence microscopy, large protein. patient’s auto-antibodies were found to react with the dermal part of the normal salt-split skin. By using a combination of immunoprecipitation and immunoblotting (IP-IB), we demonstrate They did not react with the a5(IV) deficient skin and kidney samples coming from patients with that HD1/plectin is recognized by a majority of PNP sera. Thirteen of 16 PNP sera were positive X–linked Alport’s syndrome. The patient’s IgG antibodies bound a unique 185 kDa antigen for HD1/plectin compared with none of 43 control sera (11 pemphigus vulgaris, 11 pemphigus comigrating with a5(IV) as identified by a specific antibody. Eluted antibodies from the 185 kDa foliaceus, 11 bullous pemphigoid, and 10 normal individuals). To validate our IP-IB method, we band stained the dermal part of salt-split skin confirming antibasement membrane reactivity of simultaneously examined the PNP sera for anti-DPI and anti-DPII antibodies. Thirteen of 16 IgG auto-antibodies. They further reacted with a fusion protein corresponding to the NC1 domain PNP sera could immunoprecipitate DPI and eight of 16 PNP sera could immunoprecipitate DPII. of a5(IV). This study further provides evidence for an important role of a5(IV) in physiological One PNP sera detected HD1/plectin but not DPI, and one PNP sera detected DPI but not stability of the dermal–epidermal junction. HD1/plectin. Our data strongly suggest that HD1/plectin is another autoimmune target of PNP and, together with the recent finding that desmoglein (Dsg) 3 and Dsg1 are cell surface target antigens in PNP, confirms that PNP is an autoimmune disease against desmoglein and plakin family molecules. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 533

061 062 Proteinase/Proteinase Inhibitor Imbalance in Experimental Bullous Pemphigoid Molecular Genetics of Severe Junctional Epidermolysis Bullosa Undergoing Spontaneous Ameli- Z. Liu, X. Zhou, R. Chen, S. D. Shapiro, R. M. Senior and L. A. Diaz oration Department of Dermatology, Medical College of Wisconsin; VA Medical Center, Milwaukee, Y. Gache, S. Chavanas, C. Bodemer,* C. Prost,† J. Uitto,‡ J. P. Ortonne and G. Meneguzzi Wisconsin; and Department of Internal Medicine, Washington University, St. Louis, Missouri Inserm U385, Nice, France; *Necker, †St Louis Hospitals, Paris, France; ‡Jefferson Medical Bullous pemphigoid (BP) is an autoimmune disease characterized by subepidermal blisters and College, Philadelphia, Pennsylvania autoantibodies against two hemidesmosomal proteins – BP230 and BP180. Further, the subepider- General improvement of the epithelial lesions with aging has been reported in some patients mal blisters in BP are associated with an inflammatory cellular infiltrate. Proteinases have been suffering from junctional epidermolysis bullosa (JEB). We studied a patient presenting a rapid implicated in the immunopathogenesis of BP. We previously showed that passive transfer of anti- evolution from severe to mild PA-JEB associated with reduced expression of integrin a6b4. BP180 IgG into neonatal mice induces a BP-like disease in the animals which dependents on C Genetic analysis of the gene (IGTB4) encoding integrin b4 revealed compound heterozygosity activation and neutrophil infiltration. Gelatinase B knockout (GelB–/–) and neutrophil elastase for a point mutation in exon 31 underlying severe PA-JEB, and a point mutation (3986–19T®A) knockout (NE–/–) mice are resistant to the pathogenic effects of anti-BP180 IgG, indicating that located in the lariat branchpoint consensus sequence of intron 31. By performing expression of these enzymes play crucial roles in this disease process. In this study we investigated the relationship ITGB4 minigenes in vitro, and RT-PCR on mRNA isolated from the patient’s keratinocytes, we between NE, GelB and the NE inhibitor, ,1-proteinase inhibitor (,1-PI), using the BP mouse demonstrated that under the influence of epigenetic cell factors, the intronic defect drives synthesis model. GelB–/– mice became susceptible to the experimental BP when reconstituted with 5 ϫ105 of either wild-type or abnormal b4 mRNA. In another patient with severe JEB associated with neutrophils from NE–/– mice or with 2 ϫ106 (but not 5 ϫ105) neutrophils from GelB–/– mice. absent expression of laminin b3, genetic analysis of gene LAMB3 revealed heterozygosity for the Similarly, anti-BP180 IgG induced blisters in NE–/– mice which were reconstituted with 5 ϫ105 nonsense mutation R635X in exon 14, and a two-base deletion (1587delAG) in exon 13 resulting neutrophils from GelB–/– mice; however, these animals remained resistant to the pathogenic in expression of mRNA with an in-frame exon skipping of exon 14. With age, a strong reduction effects of anti-BP180 IgG when reconstituted with NE–/– neutrophils at levels up to 2 ϫ106 of the blistering tendency correlated with enhanced expression of laminin-5 and improvement in cells per animal. In anti-BP180 treated animals, the levels of N˜ 1-PI in the circulation and at the number and structuration of hemidesmosomes. At the age of five, RT-PCR amplification of total skin injection sites were 3.2-and 2.6-fold higher, respectively, in GelB–/– compared with GelBϩ/ RNA purified from skin biopsies demonstrated upregulation of the internally deleted b3 mRNA. ϩ mice. Pretreatment of GelBϩ/ϩ mice with superoxide dismutase to block the production of Biochemical analysis of the patient’s keratinocytes demonstrated synthesis of functional laminin-5 reactive free radicals, which inactivate N˜ 1-PI, rendered the animals resistant to the pathogenic molecules with a shortened 130 kDa b3 chain missing the C-terminal EGF-like repeat of domain anti-BP180 IgG. Taken together, these results suggest that NE directly causes tissue damage in III, and the two cysteines of domain II thought to stabilize the heterotrimer. Our results BP by cleaving structural proteins at the dermal–epidermal junction, while GelB, in concert with demonstrate that cellular factors can reverse the effect of splice site mutations in genes associated free radicals, contributes to tissue damage in BP indirectly by inactivating N˜ 1-PI leading to an with severe EB. Identification of the mechanisms underlying reversion of the clinical phenotype imbalance between NE and N˜ 1-PI. is expected to pave the way to new therapeutic approaches of subsets of these disabling disorders.

063 064 Epithelial Innate Defense by Excreted PR-rich Peptides Involves Intracellular ‘‘Short Circuiting’’ XS106 Cells and Crude Murine Epidermal Cell Populations Contain mRNA for the IL- Y. R. Chan and R. L. Gallo 18 Receptor Department of Dermatology, Boston Children’s Hospital and Harvard Medical School, Boston, K. Campton, H. Ozawa, W. Ding, E. Miranda, A. Moesta, A. Tabaee and R. D. Granstein Massacusetts Weill College of Medicine of Cornell University, New York, New York Antimicrobial peptides are rapidly produced, rapidly diffusible components of the innate immune IL-18 is a recently described cytokine with several immunomodulatory activities including system and their presence on epithelial surfaces provides a powerful early response to microbial induction of gamma interferon by several target cells. Keratinocytes (KC) have been shown to be invasion. We studied a family of proline-arginine-rich antimicrobial peptides induced during capable of producing IL-18 and keratinocyte-derived IL-18 may play a role in cutaneous immunity. wound repair, focusing on a peptide called PR-39, because of its multiple actions in killing Therefore, we examined whether murine epidermal cells or the murine skin-derived dendritic bacteria and inducing syndecans on mammalian cells. Using a biologically active fragment of PR- cell line XS106 (the kind gift of A. Takashima) expressed mRNA for the IL-18 receptor. XS106 39, PR-39(15), we characterized the mechanism through which PR-39 affects mammalian cells. cells were derived from neonatal A/J epidermis and have many phenotypic characteristics of After radioligand binding analysis, PR-39 was found to bind NIH 3T3 cells in a saturable manner dendritic cells including the ability to present antigen. mRNA was isolated from a crude population consistent with the existence of a receptor. Like full-length PR-39, PR-39(15) interacts with the of CAF1 epidermal cells as well as from XS106 cells and reverse transcribed (RT). cDNA fragments cell membrane. By immunocytochemical assays in human microvascular endothelial cells, PR- coding for the murine IL-18 receptor were amplified from both cell types using the polymerase 39(15) was seen to rapidly enter cells and bind a number of cytoplasmic proteins further identified chain reaction (35 cycles) and previously published primers. A product of the expected size was by affinity chromatography of cytosolic fractions. PR-39(15) selectively binds recombinant SH3- amplified from both sets of cells. The identity of the product obtained from XS106 cells was containing proteins in gel shift assays and also coimmunoprecipitates the native SH3-containing confirmed by restriction enzyme analysis. Cutaneous dendritic cells were obtained from CAF1 protein, p130Cas. Treatment of endothelial cells with PR-39(15) below 30 µM alters p130 skin by floating ear skin dermis-side down on complete medium and harvesting cells that localization similar to that observed with integrin-mediated activation of the p130 signaling spontaneously migrated into the medium after 5 d of culture. These cells were treated with anti- pathway. However, higher concentrations of PR-39(15) resulted in decreasing activation. Structure- Thy 1.2 antibodies and complement to delete T cells. RNA was prepared from these cells and function analysis of PR-39 was done to elucidate mechanism of action in greater detail. Various RT-PCR performed. A product of the expected size for the IL-18 receptor was obtained. These regions within PR-39(15) are conserved among all syndecan-inducing PR-rich peptides. Seven results suggest that one or more components of the epidermis expresses the IL-18 receptor as do variants of these regions were synthesized and tested for function in antimicrobial assays, gel-shift XS106 cells and at least one population of migratory cutaneous dendritic cells. analysis with SH3 proteins, and kinetic binding analysis with NIH 3T3 lysates. Charge substitutions in the N-terminus resulted in vast differences in binding and antimicrobial activity. Internal sequence changes also altered binding and functional activity. This evidence supports a model that PR-39 affects cells by binding intracellular targets, thus modifying signaling pathways such as p130Cas. Therefore, PR-39 alters cell behaviors by a ‘‘short circuit’’ event where it interferes with normal intracellular signaling by binding SH3-containing components of these pathways.

065 066 Cutaneous Neuropeptide Induction of Keratinocyte Nerve Growth Factor Arachidonic Acid-Induced Generation of Superoxide Anion and Lipid Peroxidation Mediates C- G. J. Burbach, K. H. Kim, I. S. Song, J. Aranda, A. Kim, J. Brown,* C. A. Armstrong and J. C. Ansel JUN-N-Terminal Kinase Activation in Cultured Keratinocytes Department of Dermatology, Emory University, Atlanta, Georgia; *Dermatology Service, VA A. Meves, D. Peus and M. R. Pittelkow Medical Center, Portland, Oregon Department of Dermatology and Biochemistry & Molecular Biology, Mayo Clinic, Rochester, Min- Nerve growth factor (NGF) is an essential neurotrophic factor required for the growth and nesota maintenance of cutaneous sensory nerves. Little is currently known regarding the source and Arachidonic acid (AA) is a precursor of bioactive eicosanoids but has not been extensively regulation of NGF in the skin, although it has been reported that keratinocytes are capable of investigated for direct effects as a signaling molecule. AA has recently been shown to modulate producing this factor. In this study we test the hypothesis that cutaneous sensory nerves themselves cellular communication at several levels, including the activation of MAPK such as c-jun-N- have the capacity to directly modulate the production of keratinocyte NGF. This would have terminal kinase (JNK). We have characterized the activation of JNK in conjunction with lipid important implications for the maintenance and regeneration of the cutaneous neurosensory peroxidation (LPO) and superoxide generation by AA in human keratinocytes. LPO was rapidly system. To test this possibility we examined the ability of cutaneous neuropeptides known to be induced following treatment with AA (30–100 µM). The generation of lipid peroxides was released from sensory nerves to modulate keratinocyte NGF production. The effect of substance coordinately followed by JNK activation that closely correlated with LPO in a dose-and time- P (SP), substance K (SK) and calcitonin gene-related peptide (CGRP) on PAM-212 keratinocyte dependent manner. AA-induced superoxide generation, as measured by chemiluminescence using production was determined by northern blot, ELISA and PC-12 bioassay. SK, but not SP or lucigenin, demonstrated a significant and linear increase over time. LPO, superoxide generation CGRP, was capable of directly inducing PAM212 NGF mRNA expression, secreted protein and and JNK activation following AA treatment were all significantly inhibited by superoxide dismutase bioactivity in a dose dependent manner. Our results also indicate that PAM212 cells primarily overexpression in electroporated normal human keratinocytes. Inhibitors of AA metabolism, such express the neurokinin-2 receptor, which has high specific binding affinity for SK. This is the as norhydroguaiaretic acid or MK-886, only minimally inhibited superoxide generation and JNK first report demonstrating the induction of this key neurotrophic factor by neurosensory derived activation. These results demonstrate that AA directly, and not metabolites, is the primary inducer peptides such as SK. The regulation of epidermal NGF production by the neurosensory system of superoxide generation and JNK activation. A farnesyltransferase inhibitor, manumycin A as may have important consequences for the maintenance and regeneration of cutaneous nerves in well as a LPO inhibitor, n-propyl gallate strongly inhibited superoxide generation and JNK normal skin, during inflammation and wound healing. activation. These findings indicate that AA production by keratinocytes and other cells within epidermis may directly be involved in generation of reactive oxygen species, LPO and stress- related signaling responses in keratinocytes, a principal target cell in various dermatoses. 534 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

067 068 The Imidazoquinilones, Imiquimod (R-837) and R-842 Both Induce Functional but not UVB-Impaired Antigen Presenting Cells Generate Systemic Tolerance Via Splenic T Cells Phenotypic Maturation of Human Epidermal Langerhans Cells In vitro I. Kurimoto, H. Nakamura, S. Itami, K. Yoshikawa and J. W. Streilein R. Burns, B. Ferbel, M. A. Tomai,* R. L. Miller,* A. A. Gaspari Department of Dermatology, Osaka University School of Medicine, Osaka, Japan; Schepens Eye Department of Dermatology, University of Rochester School of Medicine and Dentistry, *3M Research Institute and Department of Dermatology, Harvard Medical School, Boston, Massa- Pharmaceuticals, St. Paul, Minnesota chusetts Imiquimod (R-837) and a more potent analog, R-842, have cytokine inducing activity in cultured Exposure of murine skin to acute, low dose UVB radiation (UVR) causes profound changes in human mononuclear cells. To determine whether imiquimod and R-842 enhance antigen local and systemic cutaneous immunity. While the local effects are mediated primarily by TNFa presentation by human Langerhans cells to T-lymphocytes, the primary allogeneic mixed lympho- and are expressed in genetically susceptible mice, IL-10 is a mediator of the systemic suppression cyte reaction (MLR) was utilized. Langerhans cells were studied as unfractionated EC (1%–5% of contact hypersensitivity (CH) and tolerance induction after UVR. Hapten applied to an Langerhans cells) or flow cytometry sterile cell-sorted, enriched Langerhans cells (Ͼ95% Langerhans unirradiated site immediately after the last dose of UVR readily sensitizes mice, but if the same cells). Pulse incubations (6–16 h) of unfractionated EC or enriched LC suspensions with imiquimod hapten is applied 14 d later, tolerance is induced. We have conducted experiments to determine or R-842 (0.05–5.0 µg per ml of culture medium) reproducibly enhanced antigen presentation (i) whether antigen presenting cells (APC) deficit is responsible for this failure, and if so (ii) (30%–400% increase above control Langerhans cells) in an MLR to resting, purifed T-lymphocytes. whether the deficit resides in skin, lymph nodes or spleen. When splenectomy was performed 7 To determine whether imiquimod or R-842 treatment of Langerhans cells affected lymphokine d before UVR, DNFB applied to the skin failed to induce tolerance. Similarly, IL-10 failed to profiles in the primary MLR, an ELISA was used to study IL-4 and IFN-g secreted by T- promote hapten-specific tolerance in splenectomized mice. When hapten-derivatized, unexposed lymphocytes. IFN-g production by T-lymphocytes stimulated by imiquimod-or R-842-treated skin from UVR treated mice was grafted on to naı¨ve syngeneic mice, vigorous CH was induced. Langerhans cells was increased compared with control, untreated Langerhans cells (768 ng per ml, By contrast, adherent lymph node cells from UVR or IL-10 treated mice, when hapten-derivatized 1194 ng per ml, and 418 ng per ml of IFN-g, respectively, in a representative experiment); IL-4 and injected s.c. into naı¨ve recipients, induced tolerance. These results indicate that UVR-exposed was not detected in any supernatants. Flow cytometry analysis of control-, imiquimod-, or R- mice, as well as IL-10 treated mice, acquire APC deficit that is restricted to lymph nodes, and 842-treated Langerhans cells indicated that such antigen presenting cells were in an intermediate that these defective APC interact with T cells in the spleen to generate the regulatory cells that state of maturation (CD1aϩ, HLA-DRbrightϩ, CD40lowϩ, CD86highϩ, and CD80lowϩ). RNase mediate hapten-specific tolerance. protection assays indicated that both imiquimod and R-842 increased Langerhans cells mRNA for IL-1b, TNF-a, IL-12p40, and IL-1RA compared to control Langerhans cells. These data suggest that imiquimod or R-842 enhance Langerhans cells antigen presentation to Th1- lymphocytes without altering Langerhans cells in vitro phenotypic maturation, via a cytokine dependent-mechanism. These data are highly relevant to understanding the mechanisms of action of imiquimod (R837) on Langerhans cells and its clinical activity in treating human papilloma virus infections in vivo.

069 070 Calcitonin Gene-related Peptide Induces Human Dermal Microvascular Endothelial Cell Chemok- The LPS-Induced Expression of VCAM-1 and E-selectin is Diminished by a-Melanocyte- ine Expression and Functional Adhesion of Leukocytes to Microvascular Endothelial Cells In Vitro Stimulating Hormone (a-MSH) Both In Vitro and In Vivo J. C. Ansel, G. Burbach, I. S. Song, T. Brzoska,* T. A. Luger,* C. A. Armstrong, S. W. Caughman D. -H. Kalden, M. Fastrich, T. Scholzen, T. Brzoska, C. Sunderko¨tter and T. A. Luger and T. E. Scholzen Ludwig Boltzmann Institute of Cell Biology and Immunobiology of the Skin, Department of Department of Dermatology, Emory University Atlanta, Georgia; *Department of Dermatology, Dermatology, University of Mu¨nster, Germany. University of Mu¨nster, Germany Endothelial cells play a central role in inflammation by arranging the crosstalk between the The neuropeptide calcitonin gene-related peptide (CGRP) derived from cutaneous sensory C- circulating mononuclear blood cells und the surrounding tissue. The recruitment of leukocytes fibers is capable of regulating vasodilatation and plasma extravasation during cutaneous inflammation from the first contact through the firm adhesion and finally the extravasation is mediated by as well as of modulating biological functions of epidermal and dermal cells by activating specific adhesion molecules, e.g. E-selectin and VCAM. The neuropeptide a-melanocyte-stimulating cell surface receptors. Relatively little is known about the effects of CGRP on human dermal hormone (a-MSH) is well known as a potent inhibitor in all major forms of inflammation. microvascular endothelial cell (HDMEC) activities. In this study we address the hypothesis that Therefore we investigated the effects of a-MSH regarding the expression of adhesion molecules. HDMEC express CGRP receptors and CGRP can directly modulate endothelial cell cytokine Endothelial cells (EC) were treated with the proinflammatory stimulus LPS (100 ng per ml) alone expression and adhesion of leukocytes to HDMEC. To address this possibility HDMEC isolated and with a-MSH (10–8–10–16 M). The mRNA was isolated and the expression of VCAM and E- from human foreskins or cells of the endothelial cell line HMEC-1 were stimulated with various selectin was investigated by RT-PCR. In functional assays the adhesion of lymphocytes to LPS- concentrations of CGRP and the cytokine and chemokine repertoire was analyzed by ELISA and stimulated EC was tested. The LPS-mediated upregulation of E-selectin and VCAM mRNA Northern blotting. The presence of sensory nerves directly contacting dermal vessels in human expression was significantly inhibited upon addition of a-MSH in a dose-dependent manner. skin biopsies was demonstrated by immunofluorescence using CGRP-specific antibodies and Moreover a-MSH significantly suppressed the LPS-induced adherence of lymphocytes to EC. antibodies against the endothelial cell marker CD34. In addition, the expression of CGRP receptor Furthermore, the effect of a-MSH on the expression of adhesion molecules was investigated 1 (CGRP-R1) mRNA in HDMEC was demonstrated by RT-PCR. CGRP dose-dependently in vivo. Therefore 8 µg LPS were injected sc into ears of mice, followed by application of 25 µg (1–1000 nM) induced intracellular HDMEC cAMP accumulation and selectively upregulated a-MSH i.v. The number of vessels expressing E-selectin was evaluated immuohistochemically. mRNA and protein production of the CXC chemokines IL-8 and GROa, which are powerful LPS induced sustained expression of E-selectin, but application of a-MSH resulted in a significant chemoattractants for neutrophils and lmymphocytes. A specific inhibitor of CGRP-R1 (GCRP(8– reduction of E-selectin-positive vessels after 6 and 24 h. Thus, one reason for the anti-inflammatory 37)) was capable of antagonizing this induction. Functional in vitro adhesion assays of the neutrophil- capacity of a-MSH is based on downregulation of induced adhesion molecules. like or lymphoblastoid cell lines HL-60, JY and Molt-4 to CGRP-activated HDMEC monolayer revealed that CGRP concentration-dependently upregulated leukocyte adhesion more than 50% compared to unstimulated control cells depending on the cell line analyzed. These data suggest that CGRP derived from perivascular sensory nerves is capable of regulating leukocyte chemotaxis, adhesion and transmigration and thus may have important regulatory properties on endothelial cell functions during cutaneous neurogenic inflammation.

071 072 Expression of Apoptosis-Related Proteins in Kaposi’s Sarcoma Altered Cutaneous Inflammation in Mice Deficient in Neurokinin 1 Receptors or Neutral Endo- M. Mercader, B. Taddeo, J. R. Panella, B. J. Nickoloff and K. E. Foreman peptidase Department of Pathology and Cardinal Bernardin Cancer Center, Loyola University, Maywood, T. E. Scholzen, M. Steinhoff,* N. W. Bunnett,* T. A. Luger,† C. Armstrong and J. C. Ansel Illinois Department of Dermatology, Emory University, Atlanta, Georgia; *Department of Surgery, UCSF, Previous studies suggest cytokine production may be important in Kaposi’s Sarcoma (KS); however, San Francisco, California; †Department Dermatology University of Mu¨nster, Mu¨nster, Germany this alone cannot fully explain the KS. Currently, it is unknown if apoptosis-related proteins are Substance P (SP) is a cutaneous sensory neuron-derived neuropeptide, which exhibits a variety of involved in the pathogenesis of KS; however, we found that KS tumor cells are resistant to bioactivities in peripheral tissues including vasodilatation, promotion of cell proliferation and apoptosis when cultured in methylcellulose compared to endothelial cells (EC, the likely precursor stimulation of components of the immune system through activation of its high affinity neurokinin of KS tumor cells). To further define expression of apoptosis-related proteins in KS, we evaluated 1 receptor (NK-1R). Less is known regarding its role in cutaneous disease. The biological activities expression of antiapoptotic proteins Bcl-x, Bcl-2, A-1, and Mcl-1; and pro-apoptotic proteins of SP are tightly controlled by the local release from sensory nerves, the cell-specific expression BAK and BAX at both protein (western blot) and mRNA (RT-PCR) levels. As previously shown, of NK-1R and of the presence of the SP-degrading enzyme neutral endopeptidase (NEP). In this both EC and KS cells express the cell survival protein, Bcl-xL. Expression was increased by study we test the hypothesis that the absence of NK-1R or NEP will result in a dysregulated stimulation of EC with several cytokines know to be important in KS including IL-6, oncostatin cutaneous inflammatory response. In a mouse model for contact hypersensitivity (CHS) we M, and scatter factor, but was not affected by stimulation with IFN-g or TNF-a. Expression of compared the cutaneous inflammatory response to dinitrofluorobenzene (DNFB) in mice having Bcl-xL in primary isolates of KS tumor cells was increased by IFN-g, TNF-a, IL-6 and scatter a homologous deletion of the NK-1R gene (NK-1R–/– mice) or the NEP gene (NEP–/– mice) factor, but levels of this protein were not charged in the immortalized KS cell line, SLK. As to normal wild type (ϩ/ϩ) mice. The allergic ear swelling response to DNFB in sensitized NK- published, there was no evidence of Bcl-2 expression at either the RNA or protein level in 1R(–/–) mice was significantly reduced to 35% of that observed in wild type (ϩ/ϩ) animals. primary KS tumor cells, although Bcl-2 was readily detected in the SLK cell line and EC. BAX Histologically, NK-1R(–/–) mice had less edema and cellular inflammatory infiltrate compared to and Mcl-1 were expressed in equivalent amounts in both KS and EC, and were not significantly NK-1R(ϩ/ϩ) animals during CHS. In contrast, NEP(–/–) mice had a significantly augmented upregulated by cytokine stimulation. BAK mRNA was detected in both KS cells and EC, and CHS ear swelling response as well as more edema and cellular inflammatory infiltrate compared appeared to be downregulated on stimulation with IL-6. Of interest, SLK cells and primary KS to corresponding wild type (ϩ/ϩ) animals. Cutaneous inflammation was also measured by cells constitutively express mRNA for the cell survival protein A-1, which was not detectable in microvascular plasma extravasation, which was quantitated by Evans blue leakage from dermal EC unless stimulated with IFN-g and phorbol ester. Taken together, the data indicate that over- vessels. Sensitized NEP(–/–) mice had a highly significant increase in Evans blue leakage as expression of specific cell survival proteins and their modulation by cytokines may play an compared to sensitized wild type (ϩ/ϩ) animals at 6 h and compared to nonsensitized NEP(–/– important role in the pathogenesis of KS contributing to the apoptotic-resistant phenotype of KS ) mice challenged with DNFB at 6 h and 24 h. In addition, the ear swelling response to tumor cells. epicutaneously applied capsaicin that is known to induce neuropeptide release from sensory nerves was significantly lower in NK-1R(–/–) compared to wild type (ϩ/ϩ) mice. These results indicate that the expression and regulation of SP, NK-1R and NEP have a significant role in the regulation of cutaneous inflammatory responses and thus strongly support the role of the neurological system in inflammatory skin disease. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 535

073 074 Mice Overexpressing GM-CSF in Skin are Resistant to Induction of Tolerance by UV-Light or Contact Hypersensitivity Response in Human Monocyte Chemoattractant Protein-1 Transgenic Low Dose Application of Contact Allergens Mice G. Mu¨ller, A. Mann,* K. Breuhahn,* M. Blessing,* K. Steinbrink, T. Tu¨ting, G. Go¨llner, C. N. Mizumoto, K. Iwabuchi, H. Nakamura, M. Ato, A. Shibaki, T. Kawashima, H. Kobayashi, Szalma, A. Kandemir, J. Knop and A. H. Enk. C. Iwabuchi, K. Ogasawara, A. Ohkawara and K. Onoe´ Departments of Dermatology and *Internal Medicine, University of Mainz, Germany Department of Dermatology, School of Medicine and Section of Pathology, Institute of Immunolo- To study the effect of GM-CSF overexpression in skin, two transgenic mouse lines (GM-tgA/B) gical Science, Hokkaido University, Sapporo, Japan were developed by overexpressing GM-CSF under the control of a keratin-5 promotor. Expression Epidermal Langerhans cells belong to the dendritic cell lineage and represent the major antigen of GM-CSF is constitutive in both lines as assessed by northern blot for mRNA and by ELISA presenting cells (APC) within the skin. However, the molecular mechanisms responsible for for protein production. As determined by immunofluorescence staining and FACS analysis for Langerhans cell migration to the skin are far less defined. In the present study, to examine the MHC class II and DEC-205 expression, epidermal Langerhans cell numbers were increased 3- role of Langerhans cells and MCP-1 in DNFB-induced contact hypersensitivity response (CHR), fold in GM-tg as compared to wt controls. Also there was a significant increase in DEC-205–/ we analyzed the CHR in human MCP-1 transgenic mice (hMCP-1Tgm) that produce constitu- MHC class IIϩ cells in the epidermis. When epidermal cells from GM-tg mice were used to tively high levels of hMCP-1 in the sera under the control of the human b-actin promoter. MCP- induce proliferation in allogeneic CD4ϩ T cells, proliferative response was 2–3-fold higher when 1 is a cytokine of C-C chemokine subfamily that is chemotactic in vitro for monocytes, memory transgenic EC were used. However, when equal numbers of Langerhans cells were used, no T cells and dendritic cells. Following DNFB sensitization, enhancement of CHR was demonstrated difference in the accessory capacity of tg vs wt Langerhans cells were noticed. In in vivo contact in Tgm at various periods as compared with CHR in non-Tgm. Anti-hMCP-1 antibody sensitivity assays using TNCB as Ag, no differences in epicutaneous sensitization were observed. significantly inhibited DNFB-induced CHR in Tgm. To analyze mechanisms underlying the However, when low zone tolerance experiments were performed, GM-tg mice were rather enhanced CHR, mobilization of Langerhans cells was examined in Tgm and non-Tgm. The resistant to tolerance induction. While wt mice were tolerized by repetitive application of 4.5 µg number of Langerhans cells in the Tgm skin was within a normal range. However, these mice TNCB, even doses of 0.45 µg TNCB still caused sensitization in GM-tg animals. Only doses of showed an accumulation of NLDC-145ϩ cells in the draining lymph nodes (DLN) 24 h after Ͻ 0.45 µg resulted in tolerance induction in GM-tg. These results were also confirmed by analysis DNFB sensitization. When Tgm, non-Tgm and BALB/C mice were applied with FITC, the of TNCB-specific proliferation of draining lymph node cells. While wt-mice tolerized with 4.5 µg number of FITCϩNLDC-145ϩ cells was larger in Tgm DLN than that in non-Tgm DLN 24 h TNCB did not show any significant proliferation when stimulated with Ag in vitro, cells from after the FITC application. Moreover, administration of recombinant hMCP-1 to BALB/C mice GM-tg showed a strong proliferative response to Ag in vitro. To assay for tolerance induction in led an increase of FITCϩNLDC-145ϩ cells in the DLN. In addition, 12 h after application of another system, mice were tolerized by UV-B light applied on five consecutive days and FITC, Langerhans cells in the epidermal sheets of Tgm increased in size and expressed high levels consecutive application of the allergen TNCB following standard protocols. While tolerance of I-Ad as compared with those of non-Tgm. Expression of B7-1 in 24 h-cultured Langerhans induction was readily observed in wt mice, GM-tg again showed resistance to tolerance induction cells of BALB/C mice was augmented by addition of recombinant hMCP-1 in a dose-dependent as determined by ear swelling responses. After resensitization in the absence of UV-light after 14 manner. These finding suggest that MCP-1 accelerates Langerhans cell migration from epidermis d of rest, wt controls showed a signifanctly reduced swelling response as compared with GM-tg. into the DLN and up-regulates I-Ad and B7-1 expressions on the Langerhans cells, which enhances Our data indicate that GM-tg animals show enhanced cutaneous immune reactions as compared eventually CHR. with wt controls. Further studies are necessary to determine whether the increased numbers of MHC class IIϩ cells is responsible for these findings.

075 076 Angiogenic Factors Induce Sprout Angiogenesis of Human Dermal Microvascular Endothelial Alpha-Melanocyte-Stimulating (a-MSH) Hormone Induces Antifibrotic Activity In Vivo Cells in Fibrin Gel, not in Collagen Gel M. Bo¨hm,* U. Schulte,† C. Sunderko¨tter,* and T.A. Luger*† X. Feng, R. A. F. Clark, D. Galanakis and M. G. Tonnesen *Department of Dermatology and †Ludwig Boltzmann Institute for Cell Biology and Immunobiol- Departments of Dermatology, Pathology, and Medicine, School of Medicine, SUNY, Stony ogy of the Skin, University of Mu¨nster, Germany Brook, New York, and Dermatology Section, VAMC, Northport, New York Alpha-melanocyte-stimulating hormone (a-MSH) is a pigmentation factor but has also pleiotropic Endothelial cell invasion and migration and capillary tube formation are three major processes immunomodulatory activities. We previously reported that human dermal fibroblasts express involved in angiogenesis during cutaneous wound repair and tumor growth. The study of receptors for a-MSH. Using RT-PCR and an antibody raised against the melanocortin-1 receptor angiogenesis has been hindered by the lack of a quantifiable assay for sprout angiogenesis of human (MC-1R2–18), we detected MC-1R RNA expression and MC-1R antigenicity in human dermal microvascular endothelial cells (HDMEC). To investigate the mechanisms by which angiogenic fibroblasts. We and others also reported that a-MSH can modulate the activity of certain factors and ECM proteins regulate human angiogenesis, we developed an in vitro model system in transcription factors including NF-kB in a number of skin cell types. In order to assess the which HDMEC are cultured on microcarrier beads and embedded in a three dimensional human significance of these findings, we wondered if a-MSH may affect the physiological key function fibrin gel. This model mimics the environment of the wound clot and tumor stroma in vivo, rich of fibroblasts, i.e. production of extracellular matrix proteins such as collagen and degradation of in fibrin. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) the latter by matrix metalloproteinases. Thus, we utilized a mouse model to evaluate the potential strongly induced HDMEC to form capillary sprouts which invaded and migrated into the fibrin effect of a-MSH in experimental fibrosis. Newborn BALB/C mice (n ϭ 3) were injected gel and formed capillary networks. The presence of a capillary lumen was confirmed by confocal subcutaneously with transforming growth factor-b1 (TGF-b1, 800 ng) on three consecutive days. microscopy. In contrast, platelet derived growth factor-BB (PDGF-BB) and VEGF-C, a newly This treatment resulted in fibrosis of the dermis and an inflammatory infiltrate as evidenced by described lymphangiogenesis factor, were weak stimuli for sprout formation. Thus, VEGF and light microscopy of excisional biopsies taken from the injection sites. Concomitant injection of bFGF, but not PDGF or VEGF-C, appear to be important for human angiogenesis in a fibrin- TGF-b1 and a-MSH (25 µg) reduced the TGF-b1-induced fibrosis whereas a-MSH alone or rich environment. Fibrin appeared to be critical for sprout formation. If collagen gel was used mice treated with PBS did not develop fibrosis. In accordance with our in vivo findings, instead of fibrin, VEGF and bFGF only induced HDMEC to invade and migrate as individual costimulation of human dermal fibroblasts with TGF-b1 (10 ng per ml) and a-MSH (10–6 M) cells, without formation of capillary sprouts. Sprout angiogenesis recurred with addition of fibrin in vitro resulted in attenuation of the TGF-b1-induced activation of AP-1, a transcription factor to the collagen gel. Thus we propose that the presence of fibrin, as well as VEGF and bFGF, in known to be induced by TGF-b and an important regulatory element for collagen synthesis and wound clot or tumor stroma actively promotes angiogenesis during human cutaneous wound degradation. Our findings indicate that a-MSH has antifibrotic activity in vivo and may offer novel healing or tumor progression. therapeutic avenues for the treatment of fibrotic disorders.

077 078 Membrane-Type-1 Matrix Metalloproteinase Targeting in Melanoma Cell Invasion: Identification Interferon-g Inhibits Expression of Collagenase-3 (MMP-13) by Squamous Cell Carcinoma Cells of a 20-kDa Cleavage Product via ERK1,2 Mitogen-Activated Protein Kinase K. Lehti, H. Valtanen, S. Wickstro¨m, J. Lohi and J. Keski-Oja R. Ala-aho, N. Johansson, R. Greńman, N. Fusenig and V. -M. Ka¨ha¨ri Department of Virology, The Haartman Institute, University of Helsinki, Helsinki, Finland Departments of Dermatology and Otorhinolaryngology, University of Turku, Finland; German Tumor invasion is dependent on tightly regulated proteolysis. Membrane-type matrix metallo- Cancer Research Center, Heidelberg, Germany proteinase (MT1-MMP) is a cell surface receptor and activator for gelatinase A. Relocalization of Collagenase-3 (MMP-13) is expressed by invading tumor cells in squamous cell carcinomas (SCC) MT1-MMP and gelatinase A activity to special cell surface sites has been found to induce of the head and neck and vulva, and its expression is associated with the invasion and metastasis melanoma cell invasion in vitro. The roles of cytoplasmic domain of MT1-MMP on MT1-MMP capacity of these tumors. In contrast, MMP-13 is not expressed by normal keratinocytes in vivo expression and cell invasion were analyzed in human Bowes melanoma cells by site-directed or in culture. Here, we have examined the effect of interferon-g (IFN-g) (100 U per ml) on mutagenesis. Gelatinase A was activated in all cells expressing at their surfaces wild-type or MT1- MMP-13 expression and invasion of cutaneous SCC cells (UT-SCC-7) and ras-transformed MMP forms containing a deletion (567–582, 573–582, 577–582) or point mutation (T567A, HaCaT keratinocytes (A-5), which express high basal levels of MMP-13. Treatment of cells with Y573A, S577A) in the cytoplasmic domain. Cell invasion in vitro through basement membrane IFN-g markedly (by 95%) inhibits MMP-13 mRNA abundance and production and inhibits matrices was induced by expression of wild type and truncated MT1-MMP lacking the amino transcription of MMP-13 gene in nuclear run-on assays. IFN-g also inhibits enhancement of acids 577–582. Longer C-terminal deletions decreased the induction of invasiveness. N-terminal MMP-13, collagenase-1 (MMP-1), and 92 kDa gelatinase (MMP-9) production by tumor necrosis processing of MT1-MMP to an inactive 43 kDa form correlates with gelatinase A activation in factor-a and transforming growth factor-b and inhibits invasion of cells through type I collagen fibrosarcoma cells (Lehti et al., Biochem J 334:345, 1998). Immunoblotting analyses indicated that and Matrigel. Treatment with IFN-g rapidly and transiently activates extracellular signal-regulated MT1-MMP was processed to the 43 kDa cell surface form also in melanoma cells in association kinase (ERK)1,2 and results in persistent activation of STAT1 transcription factor, detected as with the appearance of a previously unknown soluble µ20 kDa cleavage product. The cytoplasmic phosphorylation of its serine-727 and tyrosine-701. Blocking ERK1,2 cascade (Raf/MEK1,2/ domain of MT1-MMP has an important role in the regulation of cell invasion, probably by ERK1,2) by PD98059, an inhibitor of MEK1,2 activation, potently prevents inhibition of MMP- targeting MT1-MMP and generation of foci of gelatinase A activity. Inactivation of MT1-MMP 13 expression by IFN-g and also inhibits phosphorylation of serine-727 of STAT1. Infection of by cleavage of N-terminal fragment containing the active site is a rapid step of negative regulation these cells with recombinant adenovirus expressing constitutively active MEK1 results in marked required for tightly controlled proteolysis. inhibition of MMP-13 expression. These results identify IFN-g as a potent inhibitor of MMP-13 expression and invasion by SCC cells and provide evidence that ERK1,2 pathway serves as a novel inhibitory pathway in MMP-13 expression and invasion of transformed keratinocytes. 536 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

079 080 Comparison of Fibroblast Properties in Scaffold-Based and Collagen Gel Three-Dimensional Transforming Growth Factor-b Enhances Collagenase-3 (MMP-13) Expression by Human Gingival Culture Systems Fibroblasts via p38 Mitogen Activated Protein Kinase J. N. Mansbridge, E. R. Pinney, K. Liu and A. Kern L. Ravanti, L. Ha¨kkinen, H. Larjava, U. Saarialho-Kere and V. -M. Ka¨ha¨ri Advanced Tissue Sciences, Inc., La Jolla, California Department of Dermatology, University of Turku, Finland; University of British Columbia, While fibroblasts have been extensively studied in monolayer culture, their properties in three- Vancouver, BC, Canada; Department of Dermatology, Helsinki University Central Hospital, dimensional (3D) culture, which resembles their environment in vivo, are less well understood. Helsinki, Finland We compared two 3D culture systems, using collagen gel or a knitted nylon or glycollate/lactate Collagenase-3 (MMP-13) is characterized by restricted tissue specific expression and exceptionally copolymer fiber scaffold, in order to distinguish properties related to the 3D microenvironment wide substrate specificity. Expression of MMP-13 is not detected during acute cutaneous wound from those specific to the extracellular matrix. In collagen gel 3D cultures, the extracellular matrix repair, but it is expressed by fibroblasts in chronic dermal ulcers. In cultured dermal fibroblasts, was bovine collagen; whereas in 3D cultures using knitted fiber scaffolds, the extracellular matrix expression of MMP-13 is only detected when they are in contact with three-dimensional collagen. was deposited by the cells themselves. Seeded in collagen gels, two fibroblast populations could Here, we have examined the expression of MMP-13 in gingival wound repair and in gingival be distinguished based on proliferation determined by PKH dilution, 80% of the cells not dividing, fibroblasts in culture. MMP-13 expression was detected by fibroblasts in acute (5–14 d) gingival while 20% of the cells grew with a doubling time (DT) of about 2 d. In the knitted fiber scaffold wounds using immunostaining. In cultured human gingival fibroblasts MMP-13 production and 3D cultures, essentially all the cells proliferated, 28% with a DT of 1 d, 72% with a DT of 3.5 d. mRNA levels were enhanced by transforming growth factor-b (TGF-b), but not by epidermal The knitted fiber scaffold 3D cultures deposited collagen at rates up to 200 ng per 106 cells per growth factor or platelet derived growth factor-AA. Treatment of cells with TGF-b activated two day, while the collagen gel 3D cultures showed very little collagen synthesis. Cellular expression distinct mitogen-activated protein kinases (MAPK): extracellular signal-regulated kinase (ERK)1,2 of integrin a2b1, determined by flow cytometry, was upregulated 3–4-fold in collagen gel cultures in 15 min and p38 MAPK in 1 h. In contrast, Jun N-terminal kinase (JNK) was not activated. by comparison with monolayer or knitted fiber scaffold cultures. VEGF expression at the mRNA Induction of MMP-13 mRNA expression by TGF-b was inhibited by SB203580, a specific level was induced 22-fold and secretion 2–3-fold using a knitted fiber scaffold, but not in collagen inhibitor of p38 MAPK, but not by PD98059, a specific inhibitor of ERK1,2 activation. TGF-b gel cultures, by comparison with monolayers. The results indicated that the specific nature of the also increased production of stromelysin-1 (MMP-3) by gingival fibroblasts, and this induction extracellular matrix with which the cells were in contact specifically influenced cellular gene was also inhibited by SB203580. These results show that the expression of MMP-13 and its expression in a manner distinguishable from general effects of 3D culture. activator MMP-3 in gingival fibroblasts is specifically induced by TGF-b and this is dependent on the activity of p38 MAPK. This study suggests that MMP-13 and MMP-3 play a role in matrix remodeling during repair of gingival wounds and may contribute to their healing without scarring.

081 082 The a3 Laminin Subunit, a6b4 and a3b1 Integrin Coordinately Regulate Wound Healing in Interaction Among Collagenase-1, Collagen, and the Integrin a2b1 During Re-epithelialization Cultured Epithelial Cells and in the Skin J. A. Dumin, K. Dickeson, G. Goldberg, S. A. Santoro and W. C. Parks L. E. Goldfinger, S. Collawn, J. R. Couchman and J. C. R. Jones Departments of Pediatrics, Cell Biology & Physiology, Pathology, and Medicine, Washington CM Biology, North-western University Medical School, Chicago, Illinois; Department of Cell University School of Medicine, St. Louis Missouri Biology, University of Alabama, Birmingham, Alabama In response to injury, epithelial cells at the wound edge are activated to migrate, proliferate, and Previously, we demonstrated that proteolytic processing within the globular domain of the a3 differentiate to restore tissue integrity. In skin, cellular activation is characterized by induction of subunit of laminin-5 (LN5) converts LN5 from a cell motility-inducing factor to a protein matrix metalloproteinases, particularly collagenase-1 (MMP-1), and integrins, such as the a5 and complex which can trigger the formation of hemidesmosomes, cell-matrix attachment sites found av subunits, in migrating keratinocytes. Our in vivo and in vitro studies have demonstrated that in keratinocytes (J Cell Biol, 141:255–265, 1998). We prepared a monoclonal antibody (12C4) expression of MMP-1 is selectively induced by contact with type I collagen and that the catalytic against the C-terminus of the globular domain of the a3 laminin subunit. This epitope is lost from activity of this proteinase is necessary and sufficient for migration across dermal collagen. These the a3 subunit during proteolytic processing. Antibody 12C4 stains the matrix of keratinocytes data indicate that precise contact with dermal collagen mediates keratinocyte activation in the which fail to process the a3 subunit of LN5, but fails to recognize the matrix of confluent cultures wounded epidermis and suggest that the interaction between cell surface receptors and collagen of MCF–10 A cells, which process their a3 laminin subunit. In subconfluent populations of MCF– is required for MMP-1 induction. Indeed, treatment with antibodies that specifically block a2b1 10 A cells, 12C4 only stains matrix deposited at the outer edges of cell colonies. Integrin a3b1 inhibited collagen-mediated expression of MMP-1, av, and a5, as well as keratinocyte spreading, occasionally colocalizes with the staining generated by 12C4 but a6b4 integrin does not. In matrix organization, and migration. In contrast, antibodies that block a1 and a3 subunits had no wounded MCF–10 A cell cultures, the 12C4 antibody stains the matrix beneath those cells at the effect on keratinocyte behavior. Because MMP-2 (gelatinase-A) has been shown to interact with very edge of the cellular sheet that moves to cover the wound site. Similarly, in vivo, we detect avb3 on endothelial cells, we assessed if MMP-1 binds a2b1 on keratinocytes. Using recombinant expression of the unprocessed a3 laminin subunit at the leading tip of the sheet of epidermal cells I-domains and a platelet-binding assay, we have determined that pro-MMP-1 binds the I-domain which epithelializes skin wounds. In addition, functional studies, using inhibitory antibodies, of a2 and that this interaction is specific, cation-dependent, and inhibited by a2 antibodies. indicate that LN5 and its two integrin receptors (a6b4 and a3b1) are necessary for wound healing Furthermore, pro-MMP-1 coimmunoprecipitated with A2b1 from lysates of keratinocytes, and to occur in MCF–10 A cell cultures. a2b1 coimmunoprecipitated with pro-MMP-1. No other MMPs bound a2b1 and no other integrins interacted with MMP-1. Preliminary data suggests that the prodomain of pro-MMP-1 binds the a2 I-domain at a site separate from where type I collagen binds the integrin. In addition, binding or pro-MMP-1 to the a2 I-domain or to authentic a2b1 on keratinocytes converts the zymogen to an active enzyme, without cleavage of the prodomain. These data indicate that the interaction of MMP-1 with a2b1 provides a mechanism to precisely deliver this proteinase in an active form to its principal target substrate. Taken together these data suggest that the interaction of dermal collagen with epidermal a2b1 regulates expression of several wound-specific genes in keratinocytes and is essential for cell migration across the dermal matrix.

083 084 Short-term and Prolonged Hypoxia Increase Collagen Synthesis by Different Mechanisms The Cartilage Matrix Protein NCI Submodule of Type VII Collagen Binds to Fibronectin and V. Falanga and T. Yufit Constitutes a Major New Immunodominant Antigenic Epitope for EBA Autoantibodies Boston University School of Medicine, Department of Dermatology and Skin Surgery, Roger M. Chen and D. T. Woodley Williams Medical Center, Providence, Rhode Island Department of Dermatology, North-western University, Chicago, Illinois Hypoxia may play a role in the development of fibrosis, and previous work has shown increased Type VII (anchoring fibril) collagen mediates epidermal–dermal adherence and consists of a central growth and clonal expansion of fibroblasts exposed to low oxygen tension. We now have evidence collagenous triple-helix flanked by noncollagenous globular domains, NC1 and NC2. NC1 that fibroblast cultures transiently (hours to days) exposed to hypoxia (2% O2) show enhanced contains subdomains with homology to cartilage matrix protein (CMP), fibronectin-like repeats collagen synthesis and mRNA levels of a1(I) procollagen (COL1 A1). This is largely mediated by (FIN) and the A domain of von Willebrand factor (VWF-A). We demonstrated that NC1 binds TGF-b1, because it is blocked by TGF-b1 antibodies and antisense oligonucleotides and fails to to laminin 5, fibronectin and collagens (J Biol Chem 272:14516, 1997). Epidermolysis bullosa occur in fibroblasts from TGF-b1 knock-out mice. Hypoxia causes maximal stimulation of acquisita (EBA) is an acquired blistering skin disease characterized by IgG autoantibodies to type COL1 A1 luciferase-promoter constructs having 5Ј endpoints between –804 bp and –265 bp; VII collagen. We showed previously that EBA autoantibodies recognize four major antigenic further deletions to –174 bp lead to marked reduction in luciferase activity. TGF-b receptor epitopes confined to NC1 subdomains within FIN and VWF-A. In this study, we produced an expression is decreased in short-term hypoxia, suggesting that partly compensatory mechanisms additional recombinant fusion protein corresponding to the N-terminal 227 AAs (residues 1–227) are involved. In contrast to the above findings, exposure of cultures to hypoxia over multiple of NC1 and homologous to CMP. Using an enzyme-linked immunosorbent assay and by passages leads to a fibroblast phenotype characterized by high levels of COL1A1 mRNA and an immunoblot analysis, we tested sera from patients with EBA (n ϭ 20), bullous systemic lupus attenuated response to stimulation by TGF-b1. Taken together, our studies point to differential erythematosus (BSLE) (n ϭ 3), bullous pemphigoid (n ϭ 15), and normal humans (n ϭ 12). effects of acute and long-term exposure to hypoxia which, ultimately, may be responsible for Twelve of 20 EBA sera and two of three BSLE sera reacted strongly with the fusion protein in increased collagen deposition and fibrosis. Acute hypoxic exposure results in increased COL1A1 both assays. In contrast, none of the sera from healthy control subjects or patients with unrelated transcription that is mediated, in large part, by TGF-b1. Prolonged exposure to hypoxia leads to blistering skin diseases demonstrated reactivity with this new antigenic epitope. Using a solid a fibroblast phenotype of high collagen production that may be independent of TGF-b1. phase ligand binding assay, the new CMP recombinant NC1 subdomain bound specifically and strongly to fibronectin in a dose-dependent and saturable manner. Further, IgG from EBA sera containing autoantibodies to epitopes within NC1 that colocalized with the fibronectin-binding site (CMP subdomain) strongly inhibited the binding of NC1 to fibronectin. We conclude that the CMP subdomain of NC1 mediates binding of NC1 to fibronectin and constitutes a new major immunodominant antigenic epitope for EBA autoantibodies. Furthermore, perturbations of the NC1–fibronectin interaction by EBA autoantibodies may be involved in the pathogenesis of the epidermal-dermal disadherence which is a primary feature of EBA. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 537

085 086 The Role of Proa2(I)collagen Chains in the Biomechanical Function of Type I Collagen During Disruption of Integrin a6b4 Ligand Binding Through Point Mutation in Human Keratinocytes: Aging: In Vivo Studies of Oim Mouse Skin Effects upon Hemidesmosome Formation and Cell Migration C. L. Phillips, J. S. Whitsett,* B. T. Votava and J. M. Davidson* A. J. Russell, S. V. Ghaed and M. P. Marinkovich Department of Biochemistry & Child Health, University of Missouri, Columbia, Missouri; Department of Dermatology, Stanford University School of Medicine, Palo Alto, California *VAMC and Department of Pathology, Vanderbilt University, Nashville, Tennessee a6b4 integrin is a critical hemidesmosomal component. Junctional epidermolysis bullosa (JEB) Cuvalent intermolecular crosslinks between collagen molecules are believed to be principal keratinocytes lacking b4 integrin expression (due to ITGB4 PTC mutations) were retrovirally determinants of biomechanical properties of the fibrillar matrix. Oim mice (osteogenesis imperfecta transduced with b4 integrin cDNA coding for alanine substitutions in two predicted ligand binding model; homozygous for null proa2(I)collagen gene, COL1 A2) form homotrimers of a1(I), and motifs at aa136 (LBS1) or aa229, 231 and 232 (LBS2). Disruption of a6b4 binding to laminin-5 are thus unable to form a2(I)-dependent crosslinks. To evaluate the effect of a2(I) collagen chain in LBS cells was shown in three ways: (i) In the presence of inhibitory integrin b1 antibody, LBS1 deletion on type I collagen structure/function in vivo we examined the tensile properties of and LBS2 cell binding to laminin-5 was reduced to control patient cell levels whereas JEB cells collagen by incisional wound in 3 mo, 8 mo, and 2 y old oim, heterozygote (ht), and wildtype with wild type b4 cDNA (WT) were minimally affected. (ii) LBS cells showed normal levels of (wt) skin. Tensile strength of intact unwounded oim skin, 3-mo-old (a measure of organized/ migration upon collagen IV but a decreased level of migration upon laminin-5. (iii) b4 integrin crosslinked collagen) was significantly reduced relative to wt, 49.3% of the tensile strength of wt was absent extracellularly in LBS cells whereas, the entire b4 integrin chain was present with skin, suggesting that a2(I) chains play a significant role in the tensile properties of mature skin. laminin-5 in extracellular trails of WT and normal cells. Tensile properties were not significantly different between ht and wt skin. Tensile properties of While colocalization of a6b4 integrin with laminin-5, BP180 and HD1 was observed in LBS cells unwounded skin demonstrated a significant age-dependent decrease for each group; 2 y old skin which was strictly cell associated, cells containing b4 integrin ligand binding mutations combined had half the tensile strength of 3-mo-old skin. Tensile properties of 2 y old wt skin was equivalent with a truncation at aa 1328, deleting hemidesmosomal binding, showed no colocalization of to those of 3-mo-old oim skin. In contrast, we found very little differences in tensile properties laminin-5 and b4 integrin at all. This suggests two mechanisms for localization of a6b4 integrin of 14 d postincisional wounds between oim, ht, and wt, suggesting that an a2(I)-dependent with laminin-5, one through direct association with the ligand binding site, the other through an maturational process is not required in early remodeling of scar tissue. The reduced tensile indirect association via another hemidesmosomal component. The most probable candidate for properties are consistent with our preliminary findings demonstrating that oim skin collagen is this interaction is BP180 whose endodomain binds b4 integrin and whose exodomain has recently significantly more soluble than ht or wt skin collagen. In summary the a2(I) collagen chain appears been shown to bind laminin-5. to play a significant role in the tensile strength of mature skin and provides only minor contributions to the tensile properties of the collagen fibers in early wounds.

087 088 A Novel Function for Dermatan Sulfate as an Inflammatory Mediator in Skin Meta-Analysis of Risk Factors for Diabetic Foot Ulcer Healing S. F. Penc and R. L. Gallo D. J. Margolis, J. Kantor, J. Santanna, J. A. Berlin and B. L. Strom Department of Dermatology, Children’s Hospital and Harvard Medical School, Boston, Massa- University of Pennsylvania, Philadelphia, Pennsylvania chusetts Foot ulcers are a common complication of diabetes mellitus. Our primary objective was to estimate The expression of dermatan sulfate (DS), the most abundant glycosaminoglycan (GAG) in skin, is the effect of various risk factors on the probability that a diabetic insensate foot ulcer will heal. In increased in several skin diseases and wound repair. Recent findings have shown DS can influence this meta-analysis, we used individual patient-level data from the standard care arms of multiple cell signaling and communication. To study if DS functions as an inflammatory mediator, adhesion randomized clinical trials that investigated treatments for diabetic insensate foot ulcers. To estimate molecule expression was measured on cells following exposure to DS. Dermal microvascular the magnitude of the effect of a given risk factor fixed effects logistic regression odds ratios (95% endothelial cells responded to DS, but not other GAGs, with increased cell surface expression of confidence intervals) are reported. Heterogeneity was assessed using fixed effects, random effects, ICAM-1 and E-selectin. Specific DS-degrading enzymes eliminated activity. ICAM-1 levels and Bayesian models. A total of 586 patients were analyzed from five randomized clinical trials. persisted for up to 48 h after treatment with DS, whereas E-selectin levels reached maximum at The analysis revealed that those individuals with a diabetic insensate foot ulcer who healed within 12 h and returned to baseline by 24 h. To explore the mechanism of this DS-induced signaling, 20 wk of standard care treatment were more likely to have a smaller wound [0.67(0.55,0.81)], of activation of NF-kB and STAT proteins was evaluated. DS induced rapid nuclear translocation of shorter duration [0.73(0.61,0.87)], and not be Caucasian [0.64(0.43,0.96)] as compared to those NF-kB, but had no effect on STAT activation. To determine if DS directly stimulated adhesion who did not heal. The age of the patient [0.99(0.89,1.01)], Hb A1c level at the start of the study molecule expression or initiated release of autocrine inductive factors, cells were incubated in the [1.03(0.97,1.10)], and the patient’s gender [1.02(0.69,1.50)] were not associated with wound absence or presence of DS for 24 h followed by collection of media. Conditioned media was then healing. Substantial heterogeneity was not found among the studies. In summary, this is the first depleted of DS, applied to fresh cells, and surface ICAM-1 measured. While control samples did large study to investigate risk factors for diabetic insensate foot ulcer healing. Our analysis of not induce ICAM-1, DS-depleted media was active, suggesting the presence of autocrine factors patient level data showed that the use of a standard care regimen for insensate diabetic foot ulcers that induced ICAM-1. Neutralizing antibodies to TNF-a and IL-1, cytokines known to induce is most likely to be successful with patients who have younger, smaller wounds. It is our hope adhesion molecules by activating NF-kB, did not inhibit ICAM-1 induction by DS or conditioned that health-care providers will use this information to aid in their decision to use a new product, media, demonstrating that induction was independent of these cytokines. Injection of DS, but to initially use standard care, or to refer to a specialty center. not other GAGs, into mice increased the level of circulating ICAM-1, thus confirming that DS acts in vivo and will induce ICAM-1 at DS concentrations similar to those found in cutaneous wounds. These data demonstrate that DS functions as a potent inflammatory mediator in skin, and suggests a novel proinflammatory pathway that can be stimulated by release of DS from the extracellular matrix.

089 090 Increased Risk of Bullous Pemphigoid in Male and Very Old Patients. A Population-Based Study The Gatekeeper Model is Inefficient for the Delivery of Dermatologic Services on Incidence S. R. Feldman, A. B. Fleischer Jr and J. G. Chen M. Jung, W. Kippes,* G. Messer,† B. Rzany and D. Zillikens* Westwood-Squibb Center for Dermatology Research, Departments of Dermatology and Public Departments of Dermatology, Mannheim Medical School, University of Heidelberg, Universities Health Sciences, Wake Forest University School of Medicine, Winston-Salem, North Carolina of *Wu¨ rzburg and †Munich, Germany Discouraging the use of dermatologists by limiting direct access may be an inefficient use of So far, no data has been available on gender and age specific incidences for bullous pemphigoid medical resources. The purpose of this study was to determine the likelihood that patients with (BP). The aim of this study was to calculate incidences for different gender-and age-strata and to dermatologic conditions who see a primary care provider will be referred to a dermatologist Data assess risk differences between these strata. A retrospective population based cohort of BP patients on the disposition of outpatient visits to primary care physicians for one and only one dermatologic was recruited from two well-defined dermatological referral centers. In all patients, findings by diagnosis were obtained from the 1990–94 National Ambulatory Medical Care Survey. These direct and/or indirect immunofluorescence confirmed the diagnosis of BP. Incidences were data were used in an econometric model to estimate the likelihood of referral to a dermatologist calculated as newly diagnosed cases for a population of one million residents per year. Confidence for an episode of care. Of all visits for a single dermatologic diagnosis, 39% were to primary care intervals were estimated based on a Poisson distribution. For evaluation of risk between different physicians. The disposition of referral was more common for these dermatology-related visits than age and gender strata, a Poisson regression analysis was used. The total cohort comprised 94 for all office visits to primary care physicians (5.8% vs 4.5%, P Ͻ 0.001). The most frequent patients. The mean age (Ϯ standard deviation) was 79.3 Ϯ 10.2 y for women and 76.1 Ϯ 11.2 y diagnoses associated with referral were common dermatologic problems, not rare disorders. The for men. The overall incidence [95% confidence interval] for a population of one million residents number of visits per episode of care was highly dependent on the assumptions of the analysis: per year was 6.1 [2.2, 13] new cases of BP between 1989 and 1997. The highest incidence was 6.8%–18.5% for pediatricians, 8.2%–23% for family and general practitioners, and 16.6%–46.5% calculated for individuals Ͼ 90 y with 398 [360, 439] new cases of BP per one million residents for internists. The relative difficulty for primary care providers of managing skin problems is for men, and 87 [70, 108] new cases per one million residents for women. Risk [95% confidence reflected by their frequent need to refer patients with common skin problems and by the greater interval] was higher in men vs women with 1.9 [1.3, 2.9]. In addition, the risk for BP was likelihood of referral for skin disorders than for other medical conditions. The high rates of referral increased for patients above the age of 60 y. For patients Ͼ 90 y, the risk was 297 [107, 826] fold per episode of care supports the cost effectiveness of direct access to dermatologists. higher compared with the risk for patients of 60 y and younger. In conclusion, in contrast to most other autoimmune diseases, men are affected from BP almost twice as often as women. The risk for BP is rapidly increasing beyond the age of 60 y. As the structure of our population is shifting towards the aged, more people are expected to suffer from BP. Based on an average German population over 60 years of 20.34 millions (year 2015), the incidence of BP might increase to 728 [676, 782] new cases per one million residents per year in 2015. 538 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

091 092 Differences in Melanoma Stage at Diagnosis between HMO and FFS are Due to Access to Skin Cancer Primary Prevention: A Comparison Between Specialists and Generalists Health Care R. L. Hornung, N. C. Dolan, L. A. Hansen, P. R. Yarnold H. A. Pacheco, G. F. Riley,* A. L. Potosky,† D. G. Federman,‡ R. S. Kirsner Departments of Dermatology, Pediatrics, Internal Medicine, & the Institute for Health Services Department of Dermatology, University of Miami School of Medicine, Miami, Florida; *Health Research, North-western University Medical School, Chicago, Illinois Care Financing Center, Baltimore, Maryland; †National Cancer Institute, Bethesda, Maryland; The purpose of this study was to compare skin cancer knowledge and primary prevention ‡Department of Medicine, Yale University School of Medicine, New Haven, Conneticut practices among specialists (dermatologists [DERMs]) and generalists (pediatricians [PEDs], family Melanoma is the leading cause of death due to skin disease. Early diagnosis is the best way to practitioners [FPs], and general internal medicine physicians [GIMs]) through a national survey. improve prognosis. In addition to practitioner awareness, comparing systems of health care delivery We developed and pilot tested a survey instrument using 15 academically based physicians. Items may aid in improving the rate of early detection. In one study from 1985 to 1989, Health included skin cancer and photoprotection knowledge, primary prevention practices, and personal Maintenance Organizations (HMO) had earlier stage of diagnosis for certain types of cancer, attitudes and beliefs about suntanning. This survey was then sent via four separate mailings to a including melanoma, compared with age matched patients enrolled in a Fee-for-Service (FFS) delivery system. The aim of our study was to compare stage of diagnosis for melanoma as a first randomly selected national sample of 800 physicians from the American Board of Medical cancer and melanoma diagnosed after another cancer for the years 1985–93 between aged matched Specialists database (200 surveys/specialty). patients enrolled in HMO vs FFS. The overall response rate was 37%. Sixty-five percentage of the physicians were male, 35% were Using linked data from the Surveillance, Epidemiology and End Results (SEER) program with female, and 79% were in full-time practice. Knowledge about risk factors for skin cancer was Medicare enrollment files via the Health Care Financing Agency (HCFA), we compared patients quite variable, with 44% of PEDs, 78% of FPs, 67% of GIMs, compared to 95% of DERMs, 65 and older diagnosed with melanoma enrolled in Medicare-HMO and Medicare-FFS. Each correctly answering this item (pϽ 0.05). Less than 50% of physicians in all specialties responded Medicare-HMO patient was matched 2 : 1 with a Medicare-FFS patient controlling for area of correctly to a question assessing knowledge about the UV index. DERMs were more likely than residence and year of diagnosis (1479 from 1985 to 1989 and 3129 from 1990 to 1993). generalists to report skin cancer prevention practices. Ninety-seven percentage of DERMs reported Four thousand six hundred eight patients were evaluated. Mean age at diagnosis for Medicare- HMO and Medicare-FFS was 74.7 and 75.3, respectively. Sixty-two percentage were men and discussing harmful effects of UV exposure with their high risk adult patients and advising them 38% were women. Melanoma was the first cancer for 1354 Medicare-HMO patients and 2699 against sunbathing compared to 79% of primary care doctors (pϽ 0.001). Seventy-eight percentage Medicare-FFS patients. Melanoma was the second cancer for 190 Medicare-HMO patients and of DERMs reported advising all their high risk pediatric patients to use protective clothing 365 Medicare-FFS patients. Medicare-HMO enrollee’s were diagnosed with melanoma at earlier and 84% reported advising them against midday sun compared to 57% and 61% of PEDs, stages from 1985 to 1989, however, from 1990 to 1993 there was no difference in stage at respectively (pϽ 0.01). diagnosis. When melanoma was the second cancer, there was no difference in stage at diagnosis Although knowledge about what to include in skin cancer prevention counseling was fairly high between Medicare-HMO and Medicare-FFS patients. among generalists, dermatologists generally scored higher. Dermatologists also reported higher We found differences in stage at diagnosis between patients with melanoma enrolled in HMO rates of skin cancer prevention practices among high-risk patients than did primary care providers. and FFS. Our data suggests that the difference is related to access to care. HMO patients are regularly seen by their physicians with greater opportunity for screening procedures. When patients Improving access of high risk patients to dermatologists and developing interventions to enhance are already being actively followed due to a previous cancer there was no difference in stage at skin cancer preventive practices among primary care physicians are potential strategies for improving diagnosis for melanoma. The loss of difference over time may be due to increased melanoma skin cancer prevention in the future. education that has heighten the awareness for early detection.

093 094 Evaluation of Factors Associated with Skin Self-Examination Failure to Identify Changes of Melanoma is Associated with the Patient’s Age and the Histological S. A. Oliveria, P. J. Christos, A. C. Halpern, J. Fine, R. L. Barnhill and M. Berwick Type and Thickness of Melanoma Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York; Yale P. F. Hanrahan, P. Hersey and C. D’Este University Cancer Center, New Haven, Conneticut; Johns Hopkins University School of Oncology and Immunology Unit, Mater Hospital, Newcastle, NSW, Australia; Centre for Clinical Medicine, Baltimore, Maryland; and Department of Epidemiology and Biostatistics, Memorial Epidemiology and Biostatistics, Newcastle University, Newcastle, NSW, Australia Sloan-Kettering Cancer Center, New York, New York In previous studies we have shown that 75% of people presenting with thick melanoma are over Early detection and excision of thin lesions have been important in reducing mortality from the age of 50 and 66% of these are men. The present study examines the database in the Newcastle melanoma. Periodic skin self-examination (SSE) may be beneficial in identifying thin lesions. The Melanoma Unit for factors that might explain this. Data were extracted for 3435 patients seen at purpose of this study was to evaluate factors associated with SSE. The study population was the unit from 1981 to April 1997. The final sample was 2154 cases (53% males and 47% females). comprised of 650 cases with newly diagnosed melanoma (01/15/87–05/15/89) and 549 controls identified through random-digit dialing. Personal interviews were conducted to obtain information Data analysis confirmed the association of age with thickness of melanoma. Analysis of the first on demographics, phenotypic characteristics, sun practices, and screening and health behaviors. changes noticed by patients revealed that failure to identify any changes was most common in Nevus counts were performed by trained nurse interviewers. SSE practices were obtained by males Ͼ 50 y (41% compared to 34% of older females). For patients Ͻ 50 y, 30% of males and asking, ‘‘[Prior to your biopsy – for case subjects]7 did you ever (in your life) carefully examine 27% of females failed to identify changes. Failure to identify changes was associated with nodular your own skin? By this I mean actually check surfaces of your skin deliberately and purposely?’’ melanoma and site of the melanoma on the head and neck and back region. After adjustment for Information about spouse or other nonphysician examination was obtained by asking, ‘‘[Prior to histology, sex of the patient was no longer significant. The results indicate that a patient’s age, your recent biopsy – for case subjects]7 has someone other than a physician ever carefully examined nodular histological type and site of melanoma are associated with failure to detect changes. Given areas of your skin on purpose?’’ The subjects who responded positively to either question were the inherent difficulty in detection of changes at particular sites and of changes in early nodular classified as performing SSE. Logistic regression was used to model the relationship between the potential predictor variables and SSE. We included in the final model all variables with an odds melanoma, it appears unlikely that public health messages directed at self-examination by this age ratio of at least 2.0 (or 0.50) and those for which the confidence interval excluded 1 (pϽ 0.05). group will lead to earlier diagnosis. Greater emphasis should be placed on diagnosis of melanoma Female gender was identified a priori as a predictor of SSE and thus all analyses were stratified by in this age group by health professionals. gender. Age, education and marital status were identified a priori as important variables and were selected for inclusion in the final models. For males, family history of skin cancer, total nevi, skin awareness, physician exam, previous biopsy or abnormal mole, and change of diet were predictors of SSE. Phenotypic characteristics and sun practices were not important predictors of whether a male performed SSE. Use of sunscreen lotion was not associated with a greater likelihood of SSE. Important predictors of SSE in females included: hair color, total nevi, total sun exposure, skin awareness, physician exam, previous biopsy or abnormal mole, gynecologic examination, physician breast examination, breast self-examination, and use of sun screen lotion. Identifying predictors of SSE will enable health care providers to target individuals who may not be performing SSE but who are at increased risk for developing melanoma.

095 096 Reducing Risks from Malignant Melanoma Using an Interactive Multimedia Health Education The Leiden Skin Cancer Study: Results Concerning Cutaneous Malignant Melanoma Program C. Kennedy, M. Berkhout and J. N. B Bavinck C. Glazebrook, H. C. Williams, P. Garrud, A. Avery and I. Zaki Department of Dermatology, Leiden University Medical Center, Leiden, the Netherlands Divisions of Psychiatry, Dermatology and General Practice, University of Nottingham, Not- The objective of the Leiden Skin Cancer Study (LSS) is to investigate potential risk factors for tingham, UK melanoma skin cancer in the Dutch population. From January 1991 onward all cases with The aim of the study was to evaluate the effectiveness and acceptability of an interactive multimedia nonfamilial Cutaneous Malignant Melanoma (CMM) have been identified through our hospital program, ‘‘Skinsafe’’, in promoting skin protective behaviour in adults with higher-risk skin registration system. Patients at the opthalmology outpatient department without skin cancer have characteristics. In addition to informing users about ways to reduce the risk from the sun, including been invited to participate in the LSS as controls. All participants were extensively interviewed checking for early melanoma, Skinsafe gives personalised feedback of an individual’s relative risk about both occupational and recreational sun exposures, sunburns and blistering. Subsequent to of developing melanoma based on presence of risk factors such as red hair or multiple moles. In this prospective study five matched pairs of Family Practices were randomly allocated to their interview they received a total skin examination by a dermatologist. A total of 86 cases and Intervention or Control. In the intervention group, doctors and practice nurses were asked to 149 controls were enrolled. After adjustment for age and gender no significant relationships were prescribe Skinsafe to patients with higher risk characteristics. Baseline measures of melanoma demonstrated for occupational, recreational and total sun exposure. Sunburns in childhood (before knowledge and skin protective behaviours were made in 289 patients using Skinsafe and 421 the age of 13) however, was a significant risk factor for melanoma (crude OR 2.64; 1.47–4.77) patients recruited from the control practices. Subjects were followed up by postal questionnaire and also after adjustment for both age and skintype (OR 1.95; 1.04–3.68). CMM risk was at 1 mo and 6 mo to determine the impact of the program on knowledge about melanoma, significantly increased in men with more than 49 common nevi (OR 7.4; 2.50–11.76) or with attitudes to skin protection, perceived relative risk of developing melanoma and rates of skin more than one clinical atypical nevus (OR 7.12; 2.05–24.76). Women showed a significant protective behaviour, including mole checking. Ninety percentage of Skinsafe users had at least increase in CMM risk if they had skin type I or II (OR 5.43; 1.77–16.16). In our study skin one risk factor for melanoma but at baseline only 32% of these patients and 25% of high-risk controls actually rated themselves as being at greater than average risk. At 1-mo follow-up a type, sunburn before the age of 13, more than 49 common nevi, and atypical nevi were associated significantly greater proportion of high-risk Skinsafe users (51% vs 23%) now rated themselves at with a higher risk for CMM; chronic sun exposure was not directly linked to a higher risk increased risk (c2 ϭ 30.4, d.f. ϭ 1, p Ͻ 0.0001). There was also a significant group by time for melanoma. interaction for knowledge scores (F ϭ 10.65, d.f. ϭ 1, p Ͻ 0.001) with the Skinsafe group showing a greater increase in knowledge about skin protection, early signs of melanoma and risk factors for melanoma. Furthermore, Skinsafe users reported more positive attitudes to skin protection (p Ͻ 0.001) and better intentions to protect their skin in the future (p Ͻ 0.001). The program was evaluated positively by patients with 98.5% agreeing that the Family Practice was a good place to use such a program and 96% reporting that they had enjoyed using Skinsafe. Since preliminary analysis suggests that at 6 mo follow-up Skinsafe users are reporting significantly more skin-protective behaviours than controls we conclude that Skinsafe is likely to prove an effective and acceptable health-education tool in a community health setting. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 539

097 098 Smoking is Associated with Cutaneous Squamous Cell Carcinoma Association Between Severity of Atopic Eczema and Degree of Sensitization to Aeroallergens in J. N. B Bavinck, C. J. Kielich and M. T. Bastiaens School Children Department of Dermatology, Leiden University Medical Center, Leiden, The Netherlands T. Scha¨fer, J. Heinrich, M. Wjst, H. Adam, J. Ring and H. E. Wichmann The association between smoking and an increased risk of squamous cell carcinoma (SCC) of the Department of Dermatology and Allergy, Technical University; GSF Environmental and Health cervix has been well established; the association between smoking and cutaneous SCC, however, Research Center, Institute of Epidemiology, Munich, Germany is still controversial. The purpose of this study was to address the latter association. A subgroup of patients with atopic eczema exhibits aggravation through contact with aeroallergens. Using a case-control design, 71 patients with cutaneous SCC and 149 controls were included. Little is known, however, from population based studies on the association between severity of Data on smoking habits; exposure to sunlight; and skin type were collected by questionnaire, and the disease and degree of sensitization. We intended to investigate the relationship between allergic the presence of actinic keratoses; seborrheic warts; and lentigines was noted on physical examination. sensitization to aeroallergens and severity of atopic eczema in school children. On the basis of a Odds ratios were estimated by logistic regression and adjusted by entering continuous data (age) cross-sectional study of 2201 East German school children aged 5–14 y a nested case-control or ordinal data (gender; skin type) into the model. analysis on atopic eczema was performed. Atopic eczema and it’s severity was identified by a The odds ratio, adjusted for age and gender, for developing cutaneous SCC in persons with a dermatological examination. Allergen specific IgE antibodies to grass and birch pollen, cladosporium history of smoking compared with nonsmokers was 2.2 (1.04; 4.75). Similarly, significant herbarum, house dust mite (D.pter.), and cat were determined and additional information was associations with cutaneous SCC were found for well established risk factors, such as actinic obtained by a standardized questionnaire. The overall prevalence of atopic eczema was 2.5%. keratoses: 3.2 (1.48; 6.99); skin type: 2.7 (1.47; 5.15); and lentigines: 3.2 (1.25; 8.09). In addition, 45.8% of the children were sensitized to at least one allergen. Children with atopic eczema were the presence of seborrheic warts appeared to be a risk factor for cutaneous SCC: 2.4 (1.27; 4.51). sensitized significantly more often than those without the skin disease (82.5% vs 45.0%; OR ϭ Sun exposure was only weakly associated with cutaneous SCC, not reaching statistical significance: 5.76, CI 2.49–15.50). This was observed for each single allergen. The prevalence of atopic 1.3 (0.65; 2.72). eczema increased significantly with increasing RAST class (Chi-square trend test for each allergen: In this dataset, the association between smoking and an increased risk of cutaneous SCC was pϽ 0.0001). Also the prevalence of sensitization increased with the severity of the disease (Chi- established. The association between seborrheic warts and cutaneous SCC warrants further studies. square trend test for each allergen: p Ͻ 0.0001). This association was pronounced for house dust mite and cat allergen. Multiple linear regression analyses showed after adjustment for sex, age, location, and parental predisposition significant associations between the severity score of atopic eczema and concentrations of allergen specific IgE to mite (p ϭ 0.032) and cat (p ϭ 0.014). The degree of sensitization is directly associated with the severity of atopic eczema. It is speculated that a damaged skin barrier enhances early epicutaneous sensitization to aeroallergens. The specific IgE response contributes then to the severity of the disease in a dose dependent fashion. Reduction of aeroallergen exposure early in life could therefore be crucial for preventing disease prolongation.

099 100 Genetic Variation at MC1R, GSTT1, and GSTM1 May Increase Risk for Melanoma Variegate Porphyria in South America: Identification of a Novel Founder Mutation in Chilean A. Elahi, D. Lewis, C. Begg, Y. Song, P. Roy, T. Dwyer, R. Ashbolt, L. Blizzard, S. Mahabir, Patients and Its Future Implications D. Polsky, D. Coit, M. S. Brady, J. Lewis, A. Houghton, P. Myskowski, A. Halpern, J. Bolognia P. B. Cserhalmi-Friedman, J. Frank, C. Wolff,* and A. M. Christiano† and M. Berwick Department of Dermatology and †Genetics and Development, Columbia University, New York, Department Epidemiology and Biostatistics, Department Pathology, Department Surgery, and New York; *Department of Medicine, University of Chile, Santiago, Chile Department Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York; Menzies Variegate porphyria (VP) (OMIM 176200) is characterized by a partial defect in the activity of Centre, University of Tasmania, Tasmania, Australia; and Department Dermatology, Yale University, protoporphyrinogen oxidase (PPO), the seventh enzyme of the porphyrin-heme biosynthetic New Haven, Conneticut pathway. The disease is usually inherited as an autosomal dominant trait, displaying incomplete In order to refine the determination of risk for melanoma, we evaluated variation at three genetic penetrance. Clinically, VP is characterized by cutaneous symptoms on the sun exposed areas of loci: the melanocortin receptor (MC1R), an important determinant of pigmentary phenotype; the skin and by acute neurovisceral attacks which can lead to coma and death. In an effort to and the genes encoding the glutathione S-transferases theta and mu, gene products important in characterize the molecular basis of VP in South America, we studied 8 of 21 reported VP families modulating the cellular response to oxidative stress. We correlated subject genotypes with cutaneous from Chile by PCR, heteroduplex analysis, automated sequencing, restriction enzyme digestion phenotypes. We enrolled 45 subjects without a history of melanoma from Australia, 30 subjects and haplotyping analysis. We detected three different mutations, 1239delTACAC, R168H, and without a history of melanoma from the general population in Connecticut, 32 subjects with a 1330delT. Interestingly, 1239delTACAC, which results in a frameshift and a downstream premature newly diagnosed primary melanoma, and 30 subjects with multiple primary melanomas. Using termination codon, was found in patients from four unrelated families, living in different parts of PCR and restriction digests, we analyzed the MC1R genotypes for the presence of three alleles Chile. Thus, we performed haplotype analysis using five microsatellite markers which closely flank that have been associated with melanoma, Asp84Glu, Arg151Cys, and Asp294His. Also, we the PPO gene on chromosome 1q22, in the order ptel-D1S1679-D1S2705-D1S484-D1S2707- analyzed the GSTT1 and GSTM1 genes for sequence polymorphisms. There were no correlations D1S398-cen. This interval spans µ 5.2 cM on the Genethon linkage panel, indicating that these between MC1R variants or GSTT1 or GSTM1 polymorphisms and phenotypic characteristics. five markers and the PPO gene are tightly linked. Haplotype analysis revealed the occurrence of There was also no association between presence of these variants and the development of multiple 1239delTACAC on the same chromosome 1 haplotype in 11 mutation carriers from four unrelated primary melanoma. However, subjects who were null for either the GSTT1 or GSTM1 genotypes families with VP. These findings are consistent with 1239delTACAC representing a founder and heterozygous for specific alleles at MC1R had a 6.5 times greater risk of developing melanoma mutation for VP. These findings comprise the first genetic studies of the porphyrias in South than the healthy subjects (p ϭ 0.01). While these results are based on small numbers, they suggest America, and will streamline and facilitate the elucidation of the molecular basis of VP in Chile that genetic susceptibility at multiple loci may be important to the development of melanoma. as well as in its neighboring countries Argentina, Peru and Bolivia by first searching for the mutation 1239delTACAC.

101 102 Transgenic Mice Expressing Stem Cell Factor in Basal Keratinocytes Develop Postinflammatory Cytoplasmic Dynein Participates in Retrograde Melanosomal Transport Hyperpigmentation in Response to Irritant and Allergic Contactants H. Byers, M. Yaar, M. Eller, N. Jalbert and B. Gilchrest E. L. Carter, R. E. Tigelaar and B. J. Longley Department of Dermatology, Boston University School of Medicine, Boston, Massachusetts Departments of Dermatology, Columbia University, New York, New York, and Yale University, Cytoplasmic Dynein (CD) is a microtubule-associated retrograde-directed motor molecule for New Haven, Conneticut membrane-bound organelles. To determine if CD is expressed in melanocytes (MC), we performed Murine epidermis, unlike human epidermis, lacks melanocytes and is unpigmented. The epidermis RT-PCR using MC cDNA and primers complementary to human brain CD heavy chain. A of transgenic mice bearing a keratin 14 promoter (K14)-driven modified cDNA for stem cell PCR product of the expected molecular size was identified and confirmed as CD by sequence factor (SCF), the ligand of the kit receptor tyrosine kinase, is exceptional in that its basal analysis. Western blotting of total MC proteins reacted with an antihuman CD antibody identified keratinocytes continue to express SCF beyond the neonatal period, resulting in the maintenance the 74 kDa CD intermediate chain. To determine if CD plays a role in melanosome transport, of a population of pigment-producing melanocytes in the epidermis throughout life. To examine duplicate cultures were treated with CD antisense- or sense-oligonucleotides (negative control) the role of epidermal SCF in the pathogenesis of the cutaneous hyperpigmentation that and high resolution time lapse microcopy, which allows visualization of melanosomal aggregates, develops in association with many forms of acute dermatitis, we used croton oil (CO) and was performed. Antisense treated MC demonstrated a strong anterograde transport of melanosomes dinitrofluorobenzene (DNFB), respectively, to induce irritant and allergic contact dermatitis in from the cell body into the dendrites, whereas melanosomal distribution was not affected in sense K14-SCF transgenic mice and their nontransgenic littermates. Epicutaneous application of CO or treated MC. To determine whether ultraviolet irradiation modifies CD expression, MC cultures DNFB to the ears of the mice produced a significantly greater degree of ear swelling in the were exposed to increasing doses of solar simulated irradiation (5–40 mJ per cm2, metered at 285 transgenic mice compared, to their nontransgenic littermates, confirming that contact dermatitis Ϯ 5 nm), equivalent to a mild to moderate sunburn exposure for intact skin. Within 24 h, 5 and can be experimentally induced in K14-SCF transgenics and suggesting that epidermal SCF 10 mJ per cm2 induced CD protein, whereas doses 001ജ 30 mJ per cm2 were associated with contributes to the immune response in certain forms of acute dermatitis. The abdominal skin of decreased levels of CD compared to sham irradiated controls. Our data show that CD participates the mice was subsequently treated with CO or DNFB. Within 1 wk, the treated skin of the in retrograde melanosomal transport in human MC and suggest that the altered melanosomal transgenic mice became hyperpigmented and thickened; similar changes were not seen in the distribution in skin after sun exposure is due, at least in part, to decreased CD levels resulting in nontransgenic mice. Histologically, hyperpigmentation correlated with dermal melanophages and augmented anterograde transport. increased epidermal melanin, identical to the changes in human postinflammatory hyperpigmentation. We conclude that epidermal SCF is required for the development of postinflammatory hyperpigmentation in this model, and that the K14-SCF transgenic mouse represents a novel small animal model of human skin for the study of cutaneous hypersensitivity and pigmentary disorders. 540 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

103 104 The Rb/E2F Pathway in Normal versus Malignant Human Melanocytes Overexpression of h-Ski Overrides G1 Checkpoints and Increases UVB Sensitivity in Human E. Cheng, Y. Smicun and R. Halaban Melanoma Cells Department of Dermatology, Yale University School of Medicine, New Haven, Conneticut M. Haddad, D. Eling, W. Xu, E. Stavnezer and E. Medrano Compared to normal melanocytes, melanoma cell lines either overexpress hyperphosphorylated Huffington Center on Aging and Dept of Cell Biology and Dermatology, Baylor College of retinoblastoma tumor suppressor protein (Rb), or display a marked decrease in Rb expression. Medicine and VAMC, Houston, Texas, University of California at San Diego-School of Hyperphosphorylation of Rb family of proteins (pRb, p107, p130) by cyclin-dependent kinases Medicine, La Jolla, California, Department of Biochemistry, Case Western Reserve University, (CDKs), results in increased E2F-mediated transactivation of target genes and cell cycle progression. Cleveland, Ohio Using gel shift analyses and ectopic expression of plasmids encoding antisense E2F mRNA, we The cellular proto-oncogene c-ski is expressed in neural crest derived cells such as neurons, dorsal studied the role of each of the five E2F-family (E2F1–5) members of transcription factors and the root ganglia and melanocytes. Normal human melanocytes express very low levels of the Ski three Rb proteins in normal human melanocytes compared to melanoma cell lines and tumors. protein compared to malignant melanoma cells. To investigate the role of Ski in cells from Surprisingly, E2F4 was the most abundant transcription factor in proliferating normal melanocytes melanocytic origin, we generated a cell line that expresses high levels of the Ski protein. Forced existing mainly in the p107/p130 bound (inactive) form with a small fraction the in free (active) overexpression of c-Ski results in enhanced growth rate and increased apoptosis after UVB light. form. In contrast, all the melanoma cell lines and tumors expressed high levels of E2F2 and E2F4, In these cells, induction of p53 protein does not result in increased levels of p53-inducible genes and some also E2F5, that exist in the free (active) forms. Inhibition of E2F2 production in such as the Cdk-inhibitor p21Waf–1, GADD45 and Bax. Further, we demonstrate that lack of melanoma cells by transient expression of E2F2 antisense mammalian expression vector inhibited p21Waf–1 protein induction results from a transcriptional block of p53 activity. In Ski overexpressing DNA synthetic activity by 80% compared to sense-encoding plasmid. We suggest that the cells, UVB irradiation does not alter protein levels of the transcription factor E2F1, whereas it constitutive phosphorylation of Rb family members in melanoma cells induced the production of significantly reduces pRB protein levels. Altogether, these results demonstrate a novel mechanism E2F2 and E2F4, and occasionally E2F5 proteins. These free-acting transcription factors contribute whereby high levels of the protein Ski disrupt p53 and probably pRB function. We propose that to the release of melanocytes from their normal cell cycle constraints and play an important role high levels of the Ski protein may allow the melanoma cells to become more genetically unstable in malignant transformation to melanomas. without undergoing either p53 mutation or loss of heterozigosity.

105 106 Pax-3 as a Proliferation Factor for Developing Murine Melanocytes Genetically Modified Human Keratinocytes Overexpressing Hepatocyte Growth Factor Form a T. J. Hornyak, D. J. Hayes and E. B. Ziff* Hyperproliferative Epidermis Department of Dermatology, Henry Ford Health Science Center, Detroit, Michigan, and K. Hamoen and J. Morgan *Department of Biochemistry and Howard Hughes Medical Institute, New York University Center for Engineering in Medicine and Surgical Services, Massachusetts General Hospital and Medical Center, New York, New York Harvard Medical School, Shriners Burns Hospital, Boston Massachusetts Mutations in the paired homeodomain transcription factor Pax-3 are responsible for the congenital Hepatocyte growth factor (HGF) is a fibroblast-derived protein that affects the growth, motility, syndromes of pigmentation Waardenburg syndromes types 1 and 3 in humans and for variants of and differentiation of epithelial cells. To investigate the role of HGF on epidermal homeostasis, the semidominant coat color mutant Splotch (Sp) in mice. We used transgenic mice expressing we have used retroviral-mediated gene transfer to introduce the gene encoding human HGF into 001β-galactosidase from the promoter of the dopachrome tautomerase gene, an early melanoblast diploid human keratinocytes, thus causing these cells to produce a growth factor they normally marker, to characterize the effect of Pax-3 upon melanocyte development. Transgenic mice do not express. Modified cells synthesized and secreted significant levels of HGF in vitro. heterozygous for a mutation in Pax-3 (Sp/ϩ) were generated and crossed to obtain embryos Proliferation of keratinocytes overexpressing HGF was enhanced and the cells were more homozygous for the Pax-3 mutation (Sp/Sp), identifiable because of the presence of focal neural motogenic than unmodified cells under serum free conditions. The addition of an antibody that tube defects. Homozygous embryos and control littermates were examined for differences in neutralizes HGF inhibited cell growth, suggesting that HGF must be secreted by the cells to melanoblast number and migration at stages soon after migration from the neural crest. The results promote cell proliferation. To investigate the role of HGF in vivo, we grafted modified keratinocytes show that melanoblasts are reduced in number, but not absent, in Sp/Sp embryos in regions not expressing HGF onto athymic mice using acellular dermis as a template. Grafts of HGF exhibiting neural tube defects compared with control littermates. Additionally, the presence of overexpressing keratinocytes formed a hyperproliferative epidermis in vitro as well as in vivo; focal neural tube defects in regions of Sp/Sp embryos appears to block melanoblast migration. particularly at early time points after transplantation. There were significantly more cells per length Since Pax-3 expression has been noted in the dorsal neural tube, but not in melanoblasts, these of basement membrane and a significantly thicker epidermis. When analyzed for the nuclear results suggest that Pax-3 is important in melanocyte development for expanding a population of proliferation antigen Ki-67, grafts of modified epithelia showed increased numbers of proliferating cells specified as melanoblasts within the neural tube prior to migration. cells when compared to grafts of unmodified cells. This study provides additional data supporting the role of HGF as a mediator of human epidermal proliferation and suggest the possibility that the HGF mediated pathway to epidermal proliferation might play a role in the etiology of hyperproliferative skin diseases such as psoriasis. Growth factor secreting skin equivalents may also have an application in tissue engineering and woundhealing.

107 108 Increasing PKC-001β is the Rate Limiting Step in cAMP-Induced Human Melanogenesis Activation of the Melanocortin 1 Receptor and the Sensitivity of Human Melanocytes to H.-Y. Park, M. M. Murphy and B. A. Gilchrest UV Radiation Boston University School of Medicine Boston, Massachusetts M. Scott, M. Davis and Z. Abdel-Malek The cAMP-dependent pathway plays a major role in murine pigmentation. However, its role of Department of Dermatology, University of Cincinnati, Cincinnati, Ohio in human pigmentation has not been well characterized, in part because media for human Cultured normal human melanocytes respond to 001α-melanotropin (001α-MSH) with stimulation melanocytes rely on agents that raise the intracellular level of cAMP. To investigate the cAMP- of melanogenesis and proliferation. These effects are mediated by activation of the melanocortin dependent pathway in human pigmentation, cholera toxin, known to irreversibly increase the 1 receptor (MC1R), a G-protein coupled receptor. About 20 MC1R gene mutations have been cAMP level, was replaced by bovine pituitary extract (BPE, 7 ng per ml) and dibutyryl cAMP identified, and most were overexpressed in skin type I or II individuals with red hair. This (dbcAMP, 100 µM). To manipulate the cAMP level, BPE and dbcAMP were first removed for pigmentary phenotype is associated with poor tanning ability and increased risk for skin cancer. up to 7 d. Subsequent measurement of intracellular cAMP levels by ELISA showed reduction (p We have compared the dose-dependent responses of several human melanocyte cultures derived Ͻ 0.05) in cAMP level from 0.60 001Ϯ 0.1 on day 0–0.30 001Ϯ 0.05 and 0.29 001Ϯ 0.02 pg from different pigmentary phenotypes. All of these cultures, except for one which expressed very per 106 cells, on days 3 and 7, respectively, establishing that 3 day removal minimized the level low melanin content, responded to 001α-MSH with a dose-dependent increase in cAMP of cAMP. To examine the effect of increasing cAMP on human melanogenesis, BPE and dbcAMP formation, proliferation, and tyrosinase activity, beginning at 0.1 nM 001α-MSH. One culture were removed for 3 days and melanocytes were then treated with vehicle or 100 µM IBMX for had a significantly reduced response to 001α-MSH but not to forskolin, and the minimal effective 24, 48, and 72 h. Tyrosinase protein level, determined by immunoblot analysis using a monoclonal dose of 001α-MSH was increased to 10 nM in the cAMP, tyrosinase activity and proliferation antibody against human tyrosinase, was unchanged at 24 h but was induced ~2.5-fold at 48 h assays. Comparison of the effects of UV radiation (UVR) on the survival and proliferation of the through 72 h by IBMX. In contrast, tyrosinase activity, measured in paired cultures by Pomerantz above cultures revealed that they all responded with a dose-dependent growth arrest, beginning assay, was identical in vehicle and IBMX treated cultures (3819 001Ϯ 215 cpm per ug per h) at at 14 mJ per cm2, and cell death, beginning at 21 mJ per cm2. All of the cultures with a normal both 24 and 48 h; and was induced by IBMX only at 72 h to 8261 001Ϯ 115 vs 5069 001Ϯ response to 001α-MSH responded to irradiation with 21 mJ per cm2 UVR with 6%–20% cell 1215 cpm per ug per h for controls. The earlier induction of tyrosinase protein (48 h) than activity death. When treated with 001α-MSH immediately after irradiation, these cultures were stimulated (72 h) by IBMX suggests a cAMP-mediated post-translational modification of the enzyme leading to proliferate. However, the culture with reduced responsiveness to 001α-MSH underwent about β 2 to activation. PKC-001 , known to activate (phosphorylate) human tyrosinase, was thus also 50% cell death following irradiation with 21 mJ per cm UVR despite normal levels of Bcl2, and examined by immunoblot analysis. The level of PKC-001β protein was the same at 24 and 48 h failed to proliferate faster in response to 001α-MSH, following irradiation with 14 or 21 mJ per in vehicle and IBMX-treated cultures but by 72 h, IBMX had induced PKC-001β by 1.5–2-fold. cm2 UVR. Analysis of the MC1R gene in the less responsive melanocytes revealed Arg160Ser Taken together, our results suggest that cAMP increases human pigmentation by inducing tyrosinase substitution. The above results suggest that the ability of melanocytes to express functional MC1R protein within 48 h and then inducing PKC-001β within 72 h; and that PKC-001β-mediated is an important determinant of their sensitivity to UVR. activation of tyrosinase is rate limiting in cAMP-induced melanogenesis. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 541

109 110 Regulation and Activation of the 75 kDa Neurotrophin Receptor in Human Melanocytes Intracellular Transport and Localization of Human Tyrosinase after Gene Transfection Using H. Raynham, M. Yaar, H. Park, R. Dobrowsky* and B. Gilchrest Recombinant Adenovirus Vector Boston University School of Med, Boston, Massachusetts and *University of Kansas, Lawrence, K. Hirosaki, K. Jimbow, T. Yamashita and I. Wada Kansas Departments of Dermatology & Biochemistry, Sapporo Medical University School of Medicine, Melanocytes (MC) express the 75 kDa neurotrophin (NT) receptor (p75) that binds all NTs. In Sapporo, Japan the presence of Trk receptors, NTs bind both p75 and Trk and signal through Trk. However, The biogenesis of melanosomes is still largely unknown, although it is morphologically divided data suggest that even when p75 is present alone, it may be activated to signal cell survival or into four stages of maturation. We have shown recently that tyrosinase-related protein (TRP-1) apoptosis. Because in neural crest cells p75 expression is down-regulated by increased cyclic AMP is, after completion of glycosylation in the ER and Golgi with the interaction of calnexin, (cAMP) levels that occur after nerve injury, we asked whether p75 is similarly regulated in MC. deposited in the vacuoles delineated to late endosomes (Jimbow K et al. J Invest Derm 110:473 MC were stimulated with growth factor-containing medium supplemented with forskolin (50 [abstr]). It is, however, not known how tyrosinase is transported to melanosomes after exiting µ µ M) or IBMX (100 M) that increase cAMP levels. In diluent treated cells, p75 mRNA increased TGN (trans Golgi network). This study investigates the melanosome biogenesis and characterizes within 24 h but forskolin and IBMX substantially inhibited this upregulation. To determine if intracellular transport of newly synthesized tyrosinase, by transfection of human tyrosinase-gene ultraviolet (UV) irradiation that induces cutaneous injury upregulates p75, MC were sham-or 2 using recombinant adenovirus vector in SK-MEL 24 amelanotic human melanoma cells, which UV-irradiated with solar simulated light (30 mJ per cm , metered at 285 Ϯ 5 nm). After an initial did not express any tyrosinase at both mRNA and protein levels. COS 7 monkey kidney cells down-regulation within 4 h, at 24 and 48 h p75 mRNA was strongly induced in UV- versus and MRC 5 human fibroblasts were used as control tyrosinase transfectants. We found a high sham-irradiated cells. Furthermore, 48 h after irradiation, cAMP levels were Ͼ70% decreased in expression of tyrosinase protein, 65–79 kDa, in all three cell lines, MRC 5 revealing the highest UV- versus sham-irradiated cells. To investigate p75 signal transduction, we first determined by RT-PCR that trkA and trkC are not expressed in MC maintained in choleragen/TPA-free transfection-yield, followed by COS 7 and least in SK-MEL 24. However, SK-MEL 24 expressed medium. MC were incubated with 100 ng per ml nerve growth factor (NGF) or neurotrophin- the highest yield in tyrosinase protein production and the highest enzyme activity (per mg protein 3 (NT-3) for 30 min and c-Jun amino terminal kinase (JNK) activation was determined. Compared of cells). On confocal microscopy newly synthesized tyrosinase was seen as membranous in the to diluent or NT-3, NGF substantially induced phosphorylation of GST-c-Jun(1–79). Moreover, perinuclear rim and as granular in the entire cytoplasm of SK-MEL 24. Tyrosinase-positive preincubation of MC with a cyclic peptide CATDIKGKEC that binds p75 abrogated JNK granules were colocalized with Lamp 1. In the perinuclear rim, however, some of tyrosinase- activation in NGF stimulated cells. Furthermore, within 3 h p75 activation by NGF, but not by positive granules were not colocalized with Lamp 1, and some were colocalized with endosomal NT-3, lead to a 2-fold increase of intracellular ceramide. As before, ceramide increase was markers, i.e. rab 5 (early endosome) and rab 7 (late endosome), as well as adapter protein (AP)- abrogated by preincubation with the cyclic peptide. Because in cell lines, UV irradiation directly 3. Our study indicates that human tyrosinase is initially transported to early and late endosomes activates cell surface receptors such as Fas and the TNF001α receptor, we sham- or UV-irradiated and then to early melanosomes and that AP-3, not AP-1 or AP-2, is involved in the intracellular MC to determine p75 activation. Within 30 min UV-irradiation substantially induced JNK vesicular transport to melanosomes. The previously defined stage 1 melanosomes (spherical activation that was partially decreased by NT-3 pretreatment. Our data demonstrate that in MC, vacuoles) are late endosomal granules, which may simply function as transitional convergence increased cAMP levels decrease p75 upregulation and suggest that UV irradiation may induce p75 vacuoles for tyrosinase that is subsequently transported with the help of AP-3 to the ellipsoidal expression at least in part by decreasing cellular cAMP. Furthermore, p75 is specifically activated stage H melanosomes where melanin synthesis begins. by NGF, and not NT-3, indicating that in the absence of Trk A, p75 can signal alone in MC. In addition, we suggest that UV-induced activation of JNK is mediated in part through p75. The above data expand other observations indicating a role for p75 in melanocyte homeostasis.

111 112 Molecular Characterization and Immunolocalization of Muscarinic and Nicotinic Acetylcholine Sorting of TRP-1 from the TGN Occurs Via Early Endosomes and the Plasma Membrane Receptors in Human Melanocytes H. Chen, T. Salopek and K. Jimbow R. Buchli, A. Ndoye and S. Grando Division of Dermatology, University of Alberta, Edmonton, Canada, and Department of Dermato- Department of Dermatology, University of California, Davis, California logy, Sapporo Medical University, Sapporo, Japan In human epidermis, non-neuronal acetylcholine (ACh) acts as a local hormone because it mediates It has been previously suggested that tyrosinase-related protein-1 (TRP-1) may be expressed on autocrine and paracrine control of epidermal cell functions. Melanocytes (MC) are a part of the the cell surface of melanoma cells, however, it is not clear whether this is a physiological process epidermal cholinergic network because they respond to ACh and cholinergic drugs. To identify or not. In an earlier study we showed that movement of newly synthesized TRP-1 by melanoma the signaling pathway mediating ACh effects, we studied the expression of ACh receptors of the cells can be tracked when exposed to wortmannin, a potent inhibitor of phosphoinositide 3- muscarinic (mAChR) and the nicotinic (nAChR) types in human MC. Since there exist at least kinase (required for fusion between early endosomes). Our earlier observations suggested transport five molecular subtypes of mAChR, termed m1–m5, and 16 nAChR subunits able to form ACh- of TRP-1 from the TGN to a peripheral vacuolar (probable endosomal) compartment, possibly gated ion channels, we screened total melanocyte RNA using specific primers matching sequences via the plasma membrane, and then to a central perinuclear compartment with features of the late which are unique for each mAChR subtype and nAChR subunit. Since mAChRs do not have endosome. As an extension of these earlier experiments we found that treatment of MeWo introns, to distinguish between amplification of genomic DNA and cDNA, the RNA samples melanoma cells with N-ethylmaleimide (NEM) resulted in the appearance of peripherally located were pretreated with DNase I before reverse transcription. No products were amplified if reverse TRP-1 vesicles in apposition to the plasma membrane as demonstrated by immunofluorescence transcription was omitted, demonstrating the absence of contaminating genomic DNA in cDNA analysis. Subsequent exposure to wortmannin enhanced the presence of these plasma membrane samples. The PCR experiments revealed mRNA transcripts of all – the m1, m2, m3, m4, and α α α α α associated vesicles. In contrast, changes in the TRP-1 enriched compartment typically associated m5 – mAChR subtypes, and 001 1, 001 3, 001 5, 001 6, and 001 7 nAChR subunits in MC. with wortmannin treatment (i.e. appearance of a peripheral vacuolar compartment and a central The PCR products were further characterized by restriction analysis, and the results verified in macrovesicular compartment) could be prevented by NEM. Similarly, when MeWo cells were another set of experiments, using antimAChR and antinAChR antibodies corresponding to the γ AChR types amplified in PCR experiments. The specific staining of melanocyte cell surfaces was exposed to GTP001 S, vesicles in proximity to the plasma membrane were readily apparent and achieved with each antireceptor antibody used. Thus, a combination of molecular biological and were again enhanced by treatment with wortmannin. Incubation of MeWo cells with anti-TRP- immunohistochemical approaches with receptor type-specific PCR primers and antibodies, 1 antibody, revealed internalization of the exogenous antibody via peripheral vacuoles that respectively, enabled us to demonstrate the expression of both the m1, m2, m3, m4, and m5 colocalized with both endogenous TRP-1 and internalized transferrin in keeping with sorting mAChR subtypes and the 001α1, 001α3, 001α5, 001α6, and 001α7 containing nAChRs in early endosomes. Cell-surface biotinylation labelling experiments similarly confirmed the presence human MC. The newly found melanocyte AChRs may mediate the physiologic effects of paracrine of TRP-1 on the plasma membrane of melanoma cells. These results are consistent with the ACh on MC. These receptors may also provide specific targets for cholinergic drugs to achieve notion that the sorting of TRP-1 occurs via the early endosomal compartment and is expressed pharmacological control of melanogenesis in human skin. on the plasma membrane of melanoma cells.

113 114 The Effect of Oligonucleotide Size and 5001Ј Phosphate on Stimulation of Melanogenesis The Psychological Stress Induced Abnormality In Barrier Function is Mediated by Glucocorticoid M. S. Eller, I. Hadshiew, F. P. Gasparro* and B. A. Gilchrest M. Denda, T. Tsuchiya, T. Hirao, M. Takahashi, P. M. Elias* and K. R. Feingold*† Boston University School of Medicine, Boston, Massachusetts and *Thomas Jefferson University, Shiseido Research Center, Yokohama, Japan; Departments of *Dermatology and †Medicine, Philadelphia, Pennsylvania University of California, San Francisco, California We have shown that many of the cellular responses to DNA damage can be induced in vitro and Studies have been shown that psychological stress triggers or aggravates skin disease. Here, we in vivo by DNA oligonucleotides. These responses include increased melanogenesis, enhanced demonstrate that transfer of hairless mice to a different cage delays barrier recovery rates following DNA repair capacity and induction of p53 with subsequent inhibition of the cell cycle. Although barrier perturbation (65% at 3 h). Pretreatment with a phenothiazine sedative, chlorpromazine, most of the work has focused on the DNA fragment thymidine dinucleotide, pTpT, at least restored the kinetics of barrier recovery towards normal, suggesting that psychological stress is the increased melanization and induction of p53 can also be accomplished, even more dramatically, basis for this alteration in barrier homeostasis. Next, we demonstrated that plasma corticosterone with other selected oligonucleotides. The 9-mer oligonucleotide pGpApGpTpApTpGpApG is levels increase markedly (22-fold) after transfer to new cages, and that pretreatment with particularly active, typically increasing pigment content in Cloudman S91 melanoma cells 2–3 chlorpromazine blocks this increase. Additionally, systemic administration of corticosterone delays fold more than pTpT and inhibiting proliferation 20%–50% more, depending on cell type. Of barrier recovery. Finally, pretreatment with the glucocorticoid receptor antagonist, RU 486, blocks the oligonucleotides studied to date, the activity generally increases with size, although nucleotide the delay in barrier recovery produced by two different forms of psychological stress; i.e. change sequence and composition also play a role. In order to further understand the importance of of cage or immobilization. These results indicate that psychological stress stimulates increased oligonucleotide size with respect to its melanogenic activity and to closer approximate the 27–29 production of glucocorticoids, which in turn adversely effects permeability barrier homeostasis. nucleotide fragment released during excision repair in mammalian cells, we assayed a 20-mer Since derangements in barrier homeostasis can induce/exacerbate skin diseases, such as psoriasis oligonucleotide for its effect on pigmentation in S91 cells. Only in one out of three experiments and atopic dermatitis, these results provide a mechanistic link between psychological stress and did 100 µM 20-mer affect pigment content, increasing it 50% above diluent-treated control cells. In these experiments, cells similarly treated with 100 µM pTpT showed a 400%–900% increase cutaneous pathophysiology. in pigment content. Therefore, this 20-mer oligo was found to be a much less active stimulator of melanogenesis than pTpT or the 9-mer. In order to further determine which features of the oligos affect their activity, we compared the 5001Ј-phosphorylated versus nonphosphorylated form of two oligos: pTpT versus TpT and pGpApGpTpApTpGpApG versus GpApGpTpApTpGpApG. In three experiments, the stimulation of melanogenesis above controls for pTpT versus TpT was 385% vs 12.5%, 50% vs 0% and 900% vs 360%, respectively. In a separate experiment, the 5001Ј-phosphorylated 9-mer increased pigment content 1100% of control values while the nonphosphorylated form stimulated a 600% increase. These results are in agreement with published data showing uptake of DNA oligos is facilitated by the 5001Ј phosphate, and are consistent with this uptake being a variable but critical determinant of their melanogenic activity. Furthermore, confocal microscopy shows intercellular localization of ﬂuorescently tagged pTpT in treated skin cells in vitro. Determining the features that affect uptake of oligos and/or their ability to mimic DNA damage intermediates will lead to the development of more efﬁcient stimulators of these photoprotective responses, potentially reducing the risk of carcinogenesis in photodamaged skin. 542 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

115 116 Disturbed Permeability Barrier Function inTransgenic Involucrin Deficient Mice Ceramide Glucosyltransferase Expression is Regulated by Protein Kinase C in Cultured Human Ker- J.-M. Jensen, P. Diiano* and E. Proksch atinocytes Centre National Recherche Scientifique, Meudon, France; *Department of Dermatology, Univer- G. Sando, E. Howard, C. True and K. Madison sity of Kiel, Germany Marshall Dermatology Research Laboratories, Department of Dermatology, University of Iowa Involucrin, a cornified cell envelope protein, is a product of terminal differentiation in the College of Medicine, Iowa City, Iowa epidermal keratinocyte. Very recent studies have shown that covalently bound ceramides which Ceramide glucosyltransferase (CGT) is required for synthesis of the glucosylceramide (GC) are important components of cutaneous permeability barrier, are attached by ester linkage to precursors of ceramides, the predominant lipids of the epidermal permeability barrier. We previously cornified envelope proteins mainly to involucrin (JBC 273:17763–70, 1998). In addition, we reported that CGT activity, CGT mRNA, and GC increase during keratinocyte differentiation. found that the expression of major structural proteins of the epidermis, including involucrin are To probe the mechanism(s) of CGT regulation, we determined CGT activity and mRNA levels altered by permeability barrier disruption (J Invest Dermatol 111:517–523, 1998). Here, we examined during culture differentiation and in response to effectors of protein kinase C (PKC). if involucrin deficiency in a transgenic mouse model influences permeability barrier function. In Human foreskin keratinocytes were expanded in 0.07 mM keratinocyte growth medium (KGM) an involucrin deficient mouse model we investigated changes in skin barrier homeostasis. We and then either treated with PKC effectors or switched to a differentiation medium (DMEM/ assessed basal level of transepidermal water loss (TEWL), a marker of barrier function, in these Ham’s F12::3:1ϩ10% FBS (cDMEM)). CGT activity was measured in cell lysates. CGT mRNA animals and the capacity in barrier repair after tape-stripping. We found that the basal level of was analyzed by Northern blotting. TEWL was significantly increased (ϩ25%, p Ͻ 0.01, n ϭ 16) indicating a constitutive defect in Treatment of KGM cultures with 100 nM phorbol 12-myristate 13-acetate (PMA) stimulated permeability barrier function in involucrin deficient compared to wild type mice. Permeability CGT mRNA and activity as much as 70X and 8X, respectively, by 12–24 h. After 72 h, mRNA barrier repair after further experimental barrier disruption by tape-stripping (30-fold increase in decreased to ~10%, and activity to ~60%, of peak values. Induction of CGT expression was TEWL) was significantly delayed at3hto7hafter barrier disruption (p Ͻ 0.05, n ϭ 13). In specific for phorbol analogs that activate PKC. A specific inhibitor of PKC, bisindolylmaleimide summary, we showed a perturbed basal permeability barrier function and a reduced capacity in (BIM), totally inhibited TPA-induced increases in CGT mRNA and activity. 1.4 mM calcium permeability barrier repair in involucrin deficient mice. Our results suggest an important function alone did not increase CGT mRNA or activity at 24 h. In cultures switched to cDMEM, CGT of the cornified envelope protein involucrin in skin barrier homeostasis. mRNA increased as much as 90X by 24 h, then decreased to ~10% of the peak value by 72 h. CGT activity increased as much as 25X by 24 h and remained at peak values for Ͼ 6d. This increase in activity was not further stimulated by PMA and was partially inhibited by BIM. The results indicate that keratinocyte CGT expression is transcriptionally regulated via a PKC-mediated signalling pathway, as are several other genes associated with epidermal differentiation. Induction of CGT expression by cDMEM is likely mediated by additional transcriptional and post- translational processes.

117 118 Sphingolipid Activator Proteins (SAP) are Required for Epidermal Permeability Barrier Homeostasis Delayed Epidermal Barrier Repair in Patients with Sphingomyelinase Deficiency (Niemann T. Doering, W. M. Holleran, A. Potratz, G. Vielhaber, P. M. Elias, K. Suzuki and K. Sandhoff Pick Disease) Institut fu¨r Organische Chem und Biochem, Universita¨t Bonn, Germany; Department of Dermatol. M. Schmuth, F. Weber, E. Paschke,* N. Sepp and P. Fritsch U.C. San Francisco, Beiersdorf AG, Hamburg, Germany; Departments of Neurology and Department of Dermatology, University of Innsbruck, and *Department of Pediatrics, University Psychology, University of North Carolina, Chapel Hill, North Carolina of Graz, Austria Saposins (SAPs) are small molecular weight glycoproteins that activate enzyme-catalyzed degradation In normal skin ceramides are produced in the stratum corneum either by enzymatic degradation of sphingolipids; e.g. hydrolysis of glucosylceramide (GlcCer) by 001β-glucocerebrosidase, previ- of glucosylceramides to ceramide, which is known to be required for epidermal barrier homeostasis, ously shown to be critical for epidermal barrier homeostasis. Pro-saposin knockout mice (pSAP–/– or by hydrolysis of sphingomyelin. In the present study we asked whether generation of ceramide ), generated by targeted deletion/homologous recombination (Fujita et al., Human Molec Genet by the acid sphingomyelinase is also important for epidermal barrier function. Inherited mutations 5:711, 1996), display ichthyosiform dermatosis at birth. To delineate the role of saposin protein(s) of the human acid sphingomyelinase gene cause a lysosomal storage disorder known as Niemann in epidermal structure and function, and to explore the mechanism by which dermatosis forms in Pick disease. We investigated four members of a family affected by Niemann Pick disease type B. these animals, we compared the histology, ultrastructure, and lipid composition in the epidermis Sphingomyelinase activity was assessed by determining the degradation of exogenous (choline- of pSAP-deficient (–/–) with heterozygous (ϩ/–) and normal (ϩ/ϩ) littermates. Histology methyl-14C) sphingomyelin in fibroblast cultures from skin biopsies and epidermal permeability revealed moderate hyperkeratosis, but no evidence of either hyperplasia or parakeratosis in function was examined by measurement of transepidermal water loss (TEWL) with an evaporimeter pSAP–/–animals. Extensive ultrastructural abnormalities were noted with electron microscopic (Servomed, Stockholm, Sweden) under basal and stress conditions and compared to age matched analysis (RuO4), including: abnormal lamellar body (LB) contents and the persistence of abnormally control subjects. Sphingomyelinase activity was reduced to levels between 7% and 9% of control disordered arrays of secreted LB contents four-to-five layers above the stratum granulosum/stratum fibroblasts. TEWL under basal conditions was moderately increased (38% over control). The rate corneum (SC) interface, reminiscent of that seen previously in glucocerebrosidsase-deficient of recovery compared at 1, 2, 5, and 7 d following barrier disruption by tape stripping showed a Gaucher mice. Extracts from pSAP–/– SC showed markedly elevated total GlcCer, with diminished delayed regenerative response in patients whereas barrier integrity did not show any difference Cer, while the lipid-bound envelope (LBE) showed accumulation of omega(001ω)-hydroxy- between patients and controls. These findings indicate that epidermal barrier function is impaired GlcCer, with below normal 001ω-OHCers (all structures confirmed by MALDI-MS). These in patients with Niemann Pick disease providing further evidence for the role of sphingolipid results demonstrate that saposin protein(s) are critical for (i) organization of LB contents; (ii) metabolism in epidermal homeostasis. subsequent organization of lamellar membrane structures; and (iii) normal GlcCer-to-Cer processing. These studies also show that formation of the mature LBE likely occurs stepwise, with covalent attachment of 001ω-OHGlcCer preceding deglucosylation; and suggest that the ichthyosiform abnormality in pSAP-deficient animals results from alterations in SC lipid composition.

119 120 Observations on the Structure, Function, and Origin of the Lipid-Bound Envelope Topical Mevalonic Acid Stimulates De Novo Cholesterol Synthesis and Epidermal Permeability P. M. Elias, M. Fartasch, Y. Uchida and W. M. Holleran Barrier Homeostasis in Aged Mice Department of Dermatology, U.C.S.F. & Dermatology Service, V.A. Medical Center, San A. Haratake, K. Ikenaga,* N. Katoh,* H. Uchiwa, S. Hirano* and H. Yasuno* Francisco, California; and Department of Dermatology, University of Erlangen, Germany Basic Research Laboratory, Kanebo Ltd, Kanagawa, and *Department of Dermatology, Kyoto During epidermal terminal differentiation, the phospholipid-enriched plasma membrane is replaced Prefectural University of Medicine, Kyoto, Japan by a unique structure, the lipid-bound envelope (LBE). The LBE is enriched in 001ω- Extracellular lipids of the stratum corneum, which are composed of cholesterol, fatty acid and hydroxyceramides that are covalently bound to proteins on the external aspect of the cornified ceramides, are essential for the epidermal permeability barrier function. With damage to the envelope. The LBE generally is depicted in exhaustively extracted epidermis as a unilamellar barrier, a decreased capacity for epidermal lipid biosynthesis in aged epidermis results in an structure, which despite its hydrophobic content, displays no residual barrier function. Moreover, impaired repair response. Mevalonic acid is an intermediate after the rate-limiting step in cholesterol it is assumed to originate from glucosylceramide presursors, secreted with lamellar body (LB) biosynthesis, and is catalyzed by HMG-CoA reductase. In the present study, we investigated the contents. We assessed first whether processing via LB is a prerequisite for LBE formation. An effect of topical mevalonic acid on the murine epidermal permeability barrier function, comparing extensive LBE was present in: (i) five patients with Harlequin ichthyosis (HI), who exhibit an it with that of cholesterol. Topical treatment of aged mice with acetone caused a number of absence or near-absence of LB; and (ii) 10–14-day postconfluent cultured human keratinocytes dependent linear increases in TEWL, more rapidly than that in young mice. Administration of (CHK), which displays a virtual absence of LB. Moreover, these cultures contained 001µ0.15 mg mevalonic acid on the dorsal surface of aged mice enhanced resistance against disruption of the covalently bound 001ω-hydroxyceramides per mg dry weight, biochemical confirmation that an permeability barrier by subsequent acetone treatment, whereas cholesterol revealed no effect. In LBE is present. The in situ structure and function of the LBE may be more complex than that acetone damaged aged mice, administration of mevalonic acid enhanced the recovery rate of the appreciated in chloroform:methanol-extracted tissues. Hence, we used the polar solvent, absolute barrier function more than cholesterol. In young mice, neither mevalonic acid nor cholesterol pyridine, to visualize the LBE, and to disrupt the permeability barrier in intact hairless mouse had any effect on resistance against acetone damage nor the recovery rate from acetone damage. epidermis to different levels of transepidermal water loss (TEWL). Regardless of TEWL level, an In the skin of mice topically administrated mevalonic acid, a stimulation of cholesterol synthesis intact trilaminar LBE was clearly evident in micrographs from pyridine-treated samples. In contrast, and HMG-CoA reductase activity were observed, whereas no stimulation by cholesterol was the extent of residual barrier function correlated with the amount of unextracted extracellular observed. These data indicate that a topical application of mevalonic acid enhances barrier recovery lamellar membranes. These results show: (i) that the LBE in situ is a trilaminar structure; and (ii) in aged mice, which is accompanied by not only acceleration of cholesterol biosynthesis from that even as a trilaminar structure, it has no intrinsic barrier properties. Furthermore, these results mevalonic acid but also stimulation of the whole cholesterol biosynthesis via an increase of HMG- suggest that an intact lamellar body secretory pathway is not required for LBE formation, and that CoA reductase activity. an alternate pathway may deliver LBE precursors in HI and CHK. Whether such a pathway is operative in normal epidermis remains unknown. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 543

121 122 Formation of the Lipid-Bound Envelope (LBE): Insights From Glucocerebrosidase-Deficient A New Ceramide in Human Stratum Corneum Gaucher Mouse Epidermis M. E. Stewart and D. T. Downing Y. Uchida, E. Sidransky, E. I. Ginns, P. M. Elias and W. M. Holleran Marshall Research Laboratories, Department of Dermatology, University of Iowa College of Department of Dermatology, U.C.S.F. & Dermatology Service, V.A. Medical Center, San Medicine, Iowa City, Iowa Francisco, California; Kanebo Basic Research Laboratory, Odawara, Japan; and Secion on Molecular A new ceramide was discovered during the purification of ceramides from human epidermal lipid. Neurogenetics, National Institutes of Mental Health, Bethesda, Maryland The ceramides were separated by thin-layer chromatography, first in their native state, and then The lipid-bound envelope (LBE) of the stratum corneum primarily consists of omega-hydroxylated in fully acetylated form. It was found that, in its native state, the ceramide consisting of sphingosine ceramides (001ω-OHCer) covalently attached to cornified envelope proteins, and is thought to linked to an 001α-hydroxyacid overlapped a previously unrecognized ceramide. The two ceramides play a role in organizing the extracellular membrane bilayers that subserve epidermal barrier function. were easily separated after acetylation. The NMR spectrum of the new ceramide indicated that We previously showed that hydrolysis of glucosylceramides (GlcCer) by 001β-glucocerebrosidase it consisted of 6-hydroxysphingosine linked to a nonhydroxyacid. The new ceramide constituted (GlcCer’ase) generates both 001ω-OHCer and non-OHCer in the stratum corneum. In this study, about 9% of the total ceramides. we used GlcCer’ase-deficient (Gaucher) mice to determine whether deglucosylation is a prerequisite The identification of the new ceramide may clarify a recent report on the role of vitamin C (vit for normal LBE formation. Electron microscopy (RuO4) revealed not only the extensive alterations C) in the synthesis of human epidermal ceramides (Ponec et al., J Invest Dermatol 109:348–355, in lamellar bilayer structures reported previously in Gaucher mice, but also a normal-appearing 1997). Without vit C, human epidermal cell cultures fail to synthesize some of the more polar, LBE. Subsequent lipid analysis showed an extensive accumulation of GlcCer in Gaucher mice hydroxylated ceramides. The authors suggested that vit C may be required for the synthesis of versus normal littermates, in both the extractable (i.e. unbound: 21.1 Ϯ 5.3 vs 3.3 Ϯ 0.4 µg/mg 001α-hydroxyacid moieties and/or the synthesis of hydroxylated sphingosine moieties. With dry epidermis; p Ͻ 0.01) and base-hydrolyzed (i.e. covalently bound: 2.2 Ϯ 0.4 vs undetectable) knowledge of the new ceramide, their results can be interpreted as suggesting a requirement for lipid fractions. Correspondingly, the level of unbound Cer in Gaucher epidermis was significantly vit C only in the synthesis of the three ceramides containing 001α-hydroxyacid moieties. The decreased from normal (7.8 Ϯ 2.0 vs 21.8 Ϯ 1.0; p Ͻ 0.01), while only trace bound Cer was most-polar and the second-most-polar ceramides, which consist of an 001α-hydroxyacid combined detectable (vs normal ϭ 2.4 Ϯ 0.5). Finally, consistent with substrate accumulation in the absence with 6-hydroxy-sphingosine or phytosphingosine, respectively, were absent in vit C-deficient of GlcCer’ase activity, the amide-linked fatty acids of both the LBE-GlcCer (in Gaucher) and the cultures. A requirement of vit C for the synthesis of 001α-hydroxyacid moieties must account LBE-Cer (in normal) contained 001ω-OH fatty acids, the major chain lengths being identical (i.e. for the absence of the phytosphingosine-containing ceramide since another phytosphingosine- C30:0 and C32:0). These studies demonstrate that the absence of GlcCer’ase activity does not containing ceramide is unaffected. The third-most-polar band, which contains the mixture of the preclude the covalent attachment of 001ω-OHGlcCer to cornified envelope protein(s), and further new ceramide and the ceramide consisting of 001α-hydroxyacid with sphingosine, is reduced,but suggest that deglucosylation normally may occur subsequent to covalent attachment. not eliminated in vit C deficiency. This is the expected result if 001α-hydroxyacid moieties, but not 6-hydroxysphingosine moieties, require vit C.

123 124 A Specific Inhibitor of Ceramide Glucosyltransferase Alters Lamellar Granule Assembly in Cultured Platelet-Activating Factor Acetylhydrolase II Functions as an Anti-Oxidant Phospholipase in Human Keratinocytes Human Epidermal Cells K. Madison, G. Sando, E. Howard, P. Wertz and F. Platt M. Marques, S. Marques, L. Dy, Y. Pei and J. Travers Marshall Dermatology Research Laboratories, Department of Dermatology, University of Iowa Departments of Dermatology, Pharmacology and the Herman B Wells Center for Pediatric College of Medicine, Iowa City, Iowa; and Department of Biochemistry, University of Oxford, Research, Indiana University School of Medicine, Indianapolis, Indianapolis Oxford, UK Reactive oxygen species produced in many physiological intracellular reactions can act upon Glucosylceramides (GC) synthesized in the viable epidermis and packaged in lamellar granules unsaturated fatty acyl chains in membrane phospholipids producing toxic phospholipid hydroperox- (LG) are the precursors of the stratum corneum ceramides, the major lipids of the epidermal ides and a variety of oxidized phospholipids which have been shown to induce cell damage and permeability barrier. We have previously shown that ceramide glucosyl-transferase (CGT) activity apoptosis. In addition, oxidized phosphatidylcholines have been shown to act as agonists for the is induced during keratinocyte culture differentiation and is required for GC synthesis. To study platelet-activating factor (PAF) receptor. PAF-acetylhydrolase II is a recently discovered mammalian the role of GC in LG assembly, we examined the effect of a specific inhibitor of CGT [N- enzyme with phospholipase A2-like activity through its ability to hydrolyze PAF and oxidatively butyldeoxygalactonojirimycin (NB-DGJ); J Biol Chem 269:27108–114, 1994] on keratinocyte fragmented acyl groups at the sn-2 position of phospholipids. The objective of these studies was morphology and GC content. to assess for the presence of this enzyme in epidermal cells as well as to evaluate its function. Human foreskin keratinocytes were expanded in 0.07 mM keratinocyte growth medium (KGM) Immunofluorescence studies using a specific antibody directed against PAF-AHII on human skin and then switched to a differentiation medium [DMEM/Ham’s F12::3:1 ϩ 10% FBS (cDMEM)] revealed high levels of this enzyme associated with sebaceous glands, with lesser amounts found with or without 0.45 mM NB-DGJ for up to 7d. Some cultures were labeled with a fluorescent in the epidermis. Immunoblotting studies revealed PAF-AHII protein in the keratinocyte-derived ceramide and conversion to fluorescent products determined after 30 min. Lipids from control cell lines A-431 and HaCaT. We have stably transduced human epidermal cell lines with PAF- cultures and cultures treated with NB-DGJ for 3 d and 7 d were analyzed by TLC and parallel AHII using a recombinant replication-deficient retrovirus. Over-expression of this enzyme in cultures were examined by electron microscopy. epidermal cell lines was found to protect against cell death induced by the pro-oxidative stressor Both short (2 h) and long-term (3 d and 7 d) NB-DGJ treatment inhibited by 80%–90% the tert-butyl hydroperoxide. These findings suggest that this specialized phospholipase functions to conversion of a fluorescent ceramide to GC. Culture GC content was decreased to 60% of the protect epidermal cells against oxidative damage most likely by hydrolyzing oxidized phospholipids. control values after 3 d and 7 d of NB-DGJ treatment. Electron microscopy of treated cultures revealed numerous vesicles the size of LG, but with amorphous internal contents rather than the stacked lipid membrane structures of control LG. In addition, there was a striking increase in Golgi membranes in treated cultures. These results indicate that a critical level of GC is necessary for the assembly of the internal lipid lamellae of LG and suggest that Golgi membranes may be involved in their formation.

125 126 Aged-Related Changes in the IL-1 Gene Family and their Receptors Before and After Barrier Acute Keratinocyte Damage Results in the Biosynthesis of the Lipid Mediator Platelet-Activat- Abrogation ing Factor J. Ye, C. Calhoun, K. R. Feingold, P. M. Elias and R. Ghadially C. Alapatt, D. Leung, C. Johnson, K. Clay, J. Cosgrove and J. Travers Department of Dermatology,UCSF, & Dermatology Service, V.A. Medical Center, San Franci- Departments of Dermatology and Herman B Wells Center for Pediatric Research, the Indiana sco, California University School of Medicine, Indianapolis, IN; and National Jewish Medical and Research We have shown previously that chronologically aged skin displays abnormalities in permeability Center, Denver, Colorado barrier hormeostasis which become apparent when the epidermis is stressed. Underlying the Increasing evidence suggests that the phospholipid platelet-activating factor (1-alkyl-2-acetyl- abnormal barrier homeostasis in aged epidermis is a decrease in lipid content, lipid synthesis, and glycerophosphocholine; PAF) is involved in keratinocyte biology and cutaneous inflammation. epidermal proliferation. Dysregulation of cytokine production and/or biological activity could be Our previous studies have demonstrated that human keratinocytes both synthesize PAF and express responsible for impaired signaling leading to the functional abnormalities in aging skin. Previous a functional PAF receptor (PAF-R) linked to the biosynthesis of cytokines and eicosanoids. The studies have shown increases in cytokine production (e.g. IL-1001α, TNF001α) after barrier present studies examined the ability of various physiologically relevant toxic stimuli to stimulate disruption in young epidermis. Furthermore IL-1001α is known to stimulate lipid synthesis in the biosynthesis of PAF (as measured by gas chromatography/mass spectrometry) in human HaCaT extracutaneous tissue, but little is known about the expression of the IL-1 gene family and their keratinocytes. Freezing HaCaT cells resulted in PAF biosynthesis immediately upon thawing of receptors in aged epidermis under basal conditions or after barrier perturbation. the cells. In addition, exposure of cells to high temperatures (60°C) resulted in PAF production We studied the expression of IL-1001α, IL-1ra, IL-1 type I receptors (IL-1 RI) and IL-1 type II within 5 min. The pro-oxidative stimulus tert-butyl hydroperoxide (TBH; 100 µM) also stimulated receptors (IL-1 RII) in the epidermis of 15 young and 15 aged mice before and after barrier PAF synthesis within several minutes. Finally, treatment of HaCaT keratinocytes with microgram perturbation (tape stripping and acetone treatment) using an RNase protection assay. Our results amounts of the lytic toxin, Staphylococcal Alpha Toxin, induced PAF synthesis. However, show a decrease in IL-1001α and a corresponding increase in IL-1RII in aged murine epidermis. treatment of HaCaT keratinocytes with Staphylococcal Protein A did not induce PAF biosynthesis. Following barrier perturbation similar cytokine modulations occur in the aged to those in the Addition of TBH or alpha toxin to PAF-R-negative KB cells transduced with the PAF-R resulted young, but to different degrees. Following barrier barrier perturbation the level of IL-1ra is in exaggerated calcium mobilization responses in comparison to control KB cells transduced with significantly lower in the aged, and the level of the IL-1RII is significantly higher.These results blank retrovirus. These studies indicate that a variety of physiologically relevant stimuli can trigger show that there is a selective decrease or increase in the IL-1 family mRNAs both basally and epidermal cell PAF production, and that the presence of the PAF-R can lead to enhanced calcium after barrier perturbation, that could be responsible for the diminished metabolic responses, needed signaling in response to oxidative stress or a lytic bacterial toxin. Because PAF is a potent stimulus to maintain and/or restore cutaneous barrier homeostasis in aged epidermis. for pro-inflammatory cytokine production in both keratinocytes and leukocytes, these findings provide a potential mechanism by which these toxic agents can induce cutaneous inflammation. 544 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

127 128 Altered Phosphatase Activity in Mutant PTEN Proteins Associated with Cowden Syndrome Protease Nexin-1 Expression During Development and Throughout the Hair Cycle Correlates Missense Mutations With Migration of the Dermal Papilla X. Ping, H. Zhang, F. Chen, J. Tok Celebi, V. Closson, H. Tsou and M. Peacocke Y. Liu, S. Aho, R. Paus, J. Uitto, J. Stanley and G. Cotsarelis Department of Dermatology, Columbia University, New York, New York University of Pennsylvania, Philadelphia, Pennsylvania PTEN has been identified as the susceptibility gene for two autosomal dominant inherited diseases, Protease Nexin-1 (PN-1) inhibits serine proteases such as thrombin, urokinase and tissue Cowden Syndrome (CS) and Bannayan–Zonana Syndrome (BZS). Sequence analysis of PTEN plasminogen activator, and was previously localized to the dermal papilla of rat hair follicles. We reveals homology to tensin, a protein associated with focal adhesions, and to auxillin, a cytoskeletal determined the expression pattern of the PN-1 message, using in situ hybridization, throughout protein. Functional in vitro studies show that PTEN protein is a dual specific phosphatase. To folliculogenesis and the first hair cycle of the mouse. We detected signal for PN-1 message in the further understand the functional significance of altered PTEN proteins found in CS and BZS, dermal papilla of newly forming hair follicles (2–4-d-old mice) with gradual decline of signal (6– we set up an in vitro assay system to assess the phosphatase ability of wild type and mutant 7-d-old), and no signal at full blown anagen VI (10–14-d-old). PN-1 signal was again strong recombinant PTEN proteins. An acidic artificial peptide polyGlu4Tyr1 was phosphorylated by AbI during the first spontaneous catagen stage (16–18-d-old), and then was weakly detected or not protein tyrosine kinase and used as substrate. We examined a total of 12 missense mutant proteins detected during telogen (19–25-d-old). At the onset of spontaneous anagen (24–33-d-old), PN- associated with CS or BZS. All recombinant proteins, except one, showed a significant decreased 1 signal increased again, however, by mid-anagen (35-d-old), the signal was undetectable. We phosphatase activity (Ͻ90%) when compared to the wild type protein. The G129E protein showed found these changes in PN-1 expression in both BALB/C and C57BL mice, as well as in agouti a phosphatase activity similar to the wild type PTEN protein. In addition, we also tested two mice. In contrast to previous reports in the rat, which showed a progressive increase of PN-1 nonmissense mutations (an in-frame insertion and a frameshift mutation) for their phosphatase message during anagen, and a loss of signal during catagen, we show that PN-1 is highly expressed activities. The one amino acid insertion was isolated from a CS individual with early onset breast when the dermal papilla is migrating (downward during folliculogenesis and anagen onset, and cancer, and this mutant protein also showed a decreased phosphatase activity similar to that of the upward during catagen), while we found little expression during anagen VI in the mouse. These other mutant proteins. A similar result was obtained from a truncated protein resulting from a 13 results suggest that PN-1 may protect the dermal papilla from proteases involved in tissue base pair deletion in exon 8 (frameshift mutation), as described in CS individual with bilateral remodeling during hair follicle development and cycling. breast cancer and endometrial cancer. In summary, we have shown an altered phosphatase function of recombinant mutant PTEN proteins. These data demonstrate that an intact protein phosphatase activity, at least in part, is responsible for the tumor suppressor function of the PTEN gene. Moreover, alterations in phosphatase activity are associated with the phenotypic findings of CS and BZS.

129 130 Activation of Caspase-3 is Involved in Anagen-Catagen Transition of Human Hair Follicles Juxtracrine to Autocrine Conversion of HB-EGF is Essential for the Keratinocyte Migration in T. Hibino, T. Soma, I. Horii and T. Momoi Wound Healing Shiseido Research Center, Yokohama, and National Institute of Neuroscience, Kodaira, Japan S. Tokumaru, S. Higashiyama, K. Yoshino, Y. Shirakata, N. Taniguchi and K. Hashimoto We have previously shown that apoptosis is a major driving force for the regression of human Department of Dermatology, Ehime University School of Medicine, Ehime, Japan; Department hair follicles. In situ hybridization analysis demonstrated that at least four species of caspases, of Biochemistry, Osaka University Medical School, Osaka, Japan; Kanebo Ltd, Osaka, Japan executors of apoptosis, were synthesized in outer root sheath (ORS) cells of anagen hair. In the EGF-related peptides, the mainstay of the keratinocyte growth regulatory mechanism, work in present study, we analyzed changes of apoptosis-related molecules during human hair cycle. two distinct manners, autocrine and juxtracrine, mediated by a soluble form and a membrane- Especially we focused on caspase-3, since caspase-3 locates at the downstream of the caspase anchored form, respectively. We investigated the role of juxtracrine to autocrine conversion of cascade and plays a critical role in the process of apoptosis. The use of two kinds of antibodies, EGF-related peptides in a wound healing model of cultured human keratinocytes. Wounded anti-CPP32 which recognizes both pro- and active forms, and anti-p20/17, a cleavage site- keratinocytes transiently accelerated the release of soluble EGF-related peptides 8.7-fold for the directed antibody, enabled us to determine the site of caspase activation during anagen-catagen first 24 h 94% of released EGF-related peptides was HB-EGF in the conditioned medium. Matrix transition. Human scalp tissues were obtained from plastic surgery. Each hair follicle was isolated, metalloprotease inhibitor, KBR-8301 suppressed the conversion of membrane-anchored HB-EGF fixed with paraformaldehyde and embedded in paraffin. Bcl-2 immunoreactivity (IR) was always to soluble one by inhibiting the HB-EGF processing enzyme. Addition of 1 µM of KBR-8301 detected in dermal papilla (DP) cells throughout hair cycle. In addition, DP cells never became suppressed the release of HB-EGF by 93% and decreased the keratinocyte migration activity by positive for TUNEL staining even in catagen phase, indicating that DP cells were protected from 90%, although colony growth of wounded keratinocytes mediated by juxtracrine was not inhibited. apoptosis by overexpressing Bcl-2. Outermost layer cells of ORS cells in the bulge area where Furthermore, addition of 20 pg per ml of soluble HB-EGF completely recovered the keratinocyte stem cells reside were also positive for Bcl-2. In early anagen, hair germ cells above DP cells migration. These results indicate that juxtracrine to autocrine conversion of HB-EGF is essential showed intense Bcl-2 IR. Bax staining was seen in the upper part of epithelial component in for the keratinocyte migration in wound healing. anagen hair follicles. In early catagen follicles, Bax staining was extended to the lower follicle and became strongly positive in the epithelial strand of the regressing follicles. Anti-CPP32 IR was detected in the follicular epithelial cells throughout hair cycle, but never in DP cells. Intensity of IR did not vary at any stages. On the other hand, antip20/17 IR was observed in the regressing ORS cells during early to late catagen phases, suggesting that apoptosis in catagen hair follicles is regulated by the activation but not the synthesis of caspase-3.

131 132 Neutral Endopeptidase Activity is Increased in Skin and Wounds of Patients with Diabetes Regulation of Keratinocyte Growth and Differentiation in Hyperproliferative Epidermis: M. Antezana, M. Usui, N. Gibran,* J. Larsen,† J. Ansel,‡ N. Bunnett§ and J. Olerud PPAR001α Activators Restore Tissue Homeostasis Departments of Medicine (Dermatology) and *Surgery, University of Washington, Seattle, L. Ko¨mu¨ves, K. Hanley, M.-Q. Man, M. Williams, P. Elias and K. Feingold Washington; †Department of Orthopedics, VA Medical Center, Seattle, Washington; ‡Department Departments of Dermatology, Medicine, and Pediatrics, University of California San Francisco of Dermatology, Emory University, Atlanta, Georgia; §Department of Surgery, University of School of Medicine; and Veterans Affairs Medical Center, San Francisco, California California at San Francisco, San Francisco, California We recently showed that topically applied PPAR001α activators promote epidermal differentiation Cutaneous sensory nerves mediate inflammation by the release of neuropeptides such as substance in intact adult murine skin, similar to that observed in cultured human keratinocytes and in fetal P (SP) and calcitonin gene-related peptide (CGRP). Neuropeptides stimulate plasma extravasation, skin explants. The effects of PPAR001α activators on hyperproliferative epidermis, however, are expression of adhesion molecules by endothelial cells, neutrophil infiltration, and proliferation of not known. In the present study adult male hairless mice were used to analyze the effects of keratinocytes, fibroblasts, and endothelial cells. Since each of these processes is essential for normal PPAR001α activators (clofibrate or Wy14 643) on hyperproliferative epidermis, induced by wound repair, we have proposed that neuroinflammation contributes to a normal cutaneous repeated barrier abrogation (subacute model) or by essential fatty acid deficiency (chronic model). response to injury. Neutral endopeptidase (NEP) is a cell surface enzyme that competes with cell Topical treatment with PPAR001α activators resulted in a substantial decrease of epidermal surface receptors and degrades several neuropeptides including SP and CGRP thereby terminating hyperplasia in both the subacute and in chronic hyperproliferative skin. Hyperproliferative their biological actions. We hypothesized that NEP levels would be increased in patients with epidermis was characterized by an expansion of proliferative cells expressing PCNA and keratin nonhealing wounds. In this study, we determined NEP immunolocalization and enzymatic activity 5. Following topical clofibrate treatment PCNA-and keratin 5-expressing cells were restricted to in patients with diabetic neuropathy and chronic wounds. All subjects were patients with diabetes the basal layer, similar to normal epidermis. In hyperproliferative epidermis there was decreased who underwent below the knee amputations for chronic, nonhealing ulcers. Immediately after expression of involucrin, profilaggrin-filaggrin, and loricrin as assayed by in situ hybridization and amputation, skin samples were taken from (i) the ulcer margin, (ii) unwounded skin 1 cm from immunohistochemistry. Following topical clofibrate treatment cellular staining for these mRNAs the ulcer, and (iii) from the most proximal region of the amputated limb. Skin punch biopsies and proteins increased towards normal levels. Topically applied PPAR001α activators also increased from normal volunteers were evaluated as controls. NEP localization and NEP activity were apoptosis. The present study demonstrates that topical PPAR activators have profound effects on evaluated by immunohistochemistry and fluorometric assay, respectively. Wounds and unwounded epidermal gene expression in both models of hyperproliferative skin disorders. Treatment with skin from the subjects with diabetes stained more heavily for NEP than skin from normal controls. PPAR001α activators resulted in an inhibition of cell proliferation and in promotion of epidermal In subjects with diabetes, wounds and skin samples adjacent to the wounds stained more heavily differentiation, correcting the cutaneous pathology. This study identifies PPAR001α activators as than the proximal skin samples. NEP activity of unwounded skin from the lower extremity was a novel class of potential skin therapeutic agents. 2.5 times higher in subjects with diabetes than in normal controls. In subjects with diabetes, NEP activity in the wounds and skin samples adjacent to the wounds was 4-fold greater than that of skin from the proximal leg. This study shows higher NEP expression and activity in the skin and chronic ulcers of patients with diabetes than in normal control skin. Reduction in cutaneous sensory nerves as a result of diabetic neuropathy and overexpression of NEP may contribute to impaired neuroinflammation, and thus, impaired wound healing in patients with diabetes. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 545

133 134 Induction of TR4 Orphan Receptor by Retinoic Acid in Human HaCaT Keratinocytes FXR Activators Farnesol and Juvenile Hormone III Increase Transcription of the Involucrin and S. Inui, S. Itami, A. R. Haake, L. A. Goldsmith, K. Yoshikawa and C. Chang Transglutaminase Genes and Promote Keratinocyte Differentiation Departments of Pathology and Dermatology, University of Rochester Medical Center, Rochester, K. Hanley, S. He, D. Ng, D. Bikle, M. Williams, P. Elias and K. Feingold New York; Department of Dermatology, Osaka University School of Medicine, Osaka, Japan Depts. of Dermatology, Medicine, and Pediatrics, University of California San Francisco, and Human orphan receptor TR4 can modulate the transcriptional activity of the reporter gene Veterans Affairs Medical Center, San Francisco, California containing an AGGTCA direct repeat-hormone response element. Here we studied the potential Activators of FXR, an RXR-interacting nuclear hormone receptor, have been shown to stimulate role of TR4 in human HaCaT keratinocytes. Using a chloramphenicol acetyltransferase reporter epidermal development and differentiation in utero and in fetal skin explants. In the present study gene assay, it was shown that TR4 can suppress retinoic acid (RA)-induced transactivation by we sought to determine whether a direct induction of keratinocyte differentiation might underlie 47.3% in human HaCaT keratinocytes. Electrophoretic mobility shift assay indicated that this these effects. Normal human keratinocytes (NHK) were incubated in the presence or absence of suppression may be due to TR4 competition with retinoid receptors for binding to the retinoic farnesol or juvenile hormone III (JH), metabolites in the cholesterol biosynthetic pathway, under acid response element. Western blot analysis further suggested that RA can increase the expression calcium concentrations (0.03 mM) not normally permissive for differentiation. Rates of cornified of TR4 protein in human HaCaT keratinocytes and normal human keratinocytes, indicating that envelope formation, a marker of terminal differentiation, as well as mRNA and protein levels of TR4 acts as a negative feedback modulator for RA action. Interestingly, TR4 expression is two proteins required for cornified envelope formation, involucrin and transglutaminase, were increased during differentiation of normal human keratinocytes. Together, our data suggested, for increased approximately 2–3-fold by farnesol or JH treatment. Furthermore, the effects of these the first time, that an orphan receptor, such as TR4, may play an important role in retinoid-mediated FXR activators on mRNA levels were additive with maximally stimulating calcium concentrations signaling pathway in human keratinocytes, providing a new insight into keratinocyte biology. (1.2 mM). In contrast, rates of DNA synthesis were inhibited by farnesol and JH. To determine whether the increase in mRNA levels might be due to increased gene transcription, we next transfected NHK with either a 3.7 kb fragment of the involucrin promoter, or a 2.2 kb portion of the transglutaminase promoter, coupled to luciferase reporters. Farnesol and JH stimulated involucrin and transglutaminase reporter activity approximately 2-fold (in low calcium) and 3- fold (in high calcium). Finally, results from a series of truncation and internal deletion experiments revealed a farnesol-responsive region of the involucrin gene (–2452 to –1880 bp). A single base pair mutation of the AP-1 site contained in this region (–2116 to –2110 bp) abolished farnesol responsiveness. These data show that (i) farnesol and JH induce the differentiation of NHK, in part through stimulation of involucrin and transglutaminase transcription; (ii) the involucrin gene contains a farnesol-responsive region; and (iii) the AP-1 site at –2116 to –2110 bp is required for the induction of involucrin transcription by farnesol.

135 136 Ca2ϩ Independent Myosin II Phosphorylation and Contraction in Fibroblasts Hypoxia Induced Human Keratinocyte Motility is Mediated by the PKC Pathway Modulating M. Kolodney and H. Yee, Jr the Expression of MMP-9 Division of Dermatology and Division of Digestive Diseases, Department of Medicine, UCLA E. A. O’Toole, T. A. Mustoe* and D. T. Woodley School of Medicine, Los Angeles, California Departments of Dermatology and *Plastic Surgery, North-western University, Chicago, Illinois We examined the role of cytosolic Ca2ϩ in regulation of fibroblasts force production. Dermal Hypoxia promotes keratinocyte migration on wound bed connective tissues (CT) and is a profound fibroblasts exert forces on extracellular matrix that serve to organize collagen during development, biological signal that transforms a basal keratinocyte, destined to differentiate, into a motile cell remodel granulation tissue, and contract the edges of skin wounds. Fibroblast contractility, like that is essential for reepithelialization (J Clin Invest, 1997). In this study, we examined the effect that of other nonmuscle cell types, is widely believed to be mediated through Ca2ϩ-stimulated of hypoxia on keratinocyte-derived collagenases associated with keratinocyte migration. Cells myosin II regulatory light chain (LC20) phosphorylation, similar to the contractile regulation of plated on various CT matrices under normoxic and hypoxic conditions demonstrated at least a smooth muscle. However, this hypothesis lacks conclusive experimental support. In order to 2-fold increase in the 92 kDa, type IV collagenase (MMP-9) when examined by quantitative investigate the regulation of fibroblast contraction, we utilized a novel method to quantitatively zymography, ELISA, western and northern blotting. The hypoxia-induced increase in cell motility measure isometric force generation by cultured cells. By modulating fibroblast cytosolic Ca2ϩ was inhibited by 10 µg per ml of a neutralizing antibody to MMP-9. Similar assays demonstrated 2ϩ 2ϩ concentration [Ca ]i, we tested the putative importance of [Ca ]i in fetal bovine serum (FBS) at least a 2-fold increase in tissue inhibitor of metalloproteinase (TIMP-1), an enzyme that binds 2ϩ stimulated LC20 phosphorylation and force development. Eliminating the FBS-stimulated [Ca ]i to MMP-9 and inhibits activity. Northern blotting demonstrated that TIMP-1 mRNA increased rise by loading cells with BAPTA (a Ca2ϩ chelator) only partially inhibited FBS-stimulated LC20 2-fold and 6-fold, respectively, 4 and 12 h after the cells were made hypoxic. Cells plated on phosphorylation and did not significantly alter the magnitude of FBS-stimulated isometric various CT matrices under hypoxic conditions did not demonstrate any change in MMP-I 2ϩ 2ϩ contraction. Ionomycin (a Ca ionophore) produced a larger but shorter lasting rise in [Ca ]i expression, a collagenase that is necessary for migration on collagen. The hypoxia-induced increases relative to FBS. However, ionomycin only stimulated a small and transient increase in LC20 in MMP-9 and TIMP-1 were inhibited by staurosporine and bisindolylmaleimide, inhibitors of phosphorylation and did not cause contraction. We conclude that fibroblasts differ from smooth protein kinase C (PKC), but not by inhibitors of tyrosine phosphorylation and the MAP kinase muscle in that LC20 phosphorylation and contraction are predominantly regulated independently pathway. These data provide evidence that hypoxia-induced keratinocyte migration is mediated 2ϩ of [Ca ]i. by increased cellular secretion of MMP-9 via the PKC pathway.

137 138 Requirement of Phospholipase C-001γ1 for Keratinocyte Differentiation Interleukin-1 Protects Transformed Keratinocytes from TRAIL- and CD95 L-Induced Apoptosis Z. Xie, D. D. Bikle and E. Unit via Activation of NF001κB, but not from UV-Induced Apoptosis VA Medical Center, University of California, San Francisco, California G. Kothny-Wilkes, D. Kulms, T. Luger and T. Schwarz The phospholipase C (PLC) family of enzymes play an important role in intracellular signaling Department of Dermatology, University of Mu¨nster, Mu¨nster, Germany pathways. These enzymes catalyze the hydrolysis of phosphatidyl inositol bisphosphate (PIP2), Treatment of transformed cells with TRAIL, a member of the tumor necrosis factor (TNF) family, resulting in the production of two second messengers, inositol triphosphate (IP3) and diacylglycerol results in apoptosis. Recently, we showed that interleukin-1 (IL-1) protects the transformed (DAG), in response to extracellular stimuli. Addition of calcium leads to keratinocyte differentiation keratinocyte cell line KB from TRAIL-induced apoptosis. The protective effect seems to be due and the activation of several PLC isozymes, one of which is PLC-001γ1, the predominant isoform to activation of the transcription factor NF001κB because chemical inhibitors of NF001κB in keratinocytes. However, the role of PLC-001γ1 has received little study in the regulation of activation reverse the protective effect of IL-1. Since transformed cells may escape apoptosis by keratinocyte differentiation by calcium. To address this issue, expression of PLC-001γ1 in human this mechanism we were interested whether IL-1 also protects cells from apoptosis induced by keratinocytes was blocked by transfecting cells with the antisense human PLC-001γ1 cDNA and other stimuli including CD95 ligand (CD95L) and ultraviolet light (UV). IL-1 almost completely a neomycin resistant gene, included in the constitutive expression vector pcDNA3.1(ϩ) (Invi- protected KB cells from TRAIL- and CD95L-induced apoptosis. This effect was strictly dependent trogen). The transfected cells were selected by neomycin before calcium treatment. These cells on NF001κB since KB cells transfected with a NF001κB repressor construct were not protected demonstrated a dramatic reduction in PLC-001γ1 protein level compared to empty vector- by IL-1. In contrast, apoptosis induced by UV was not only not prevented by IL-1 but even was transfected cells and a marked reduction in involucrin and transglutaminase mRNA and protein significantly enhanced. However, apoptosis induced by IL-1 plus UV was significantly reduced expression following administration of calcium. Similarly, Cotransfection of antisense PLC-001γ1 by an antibody blocking the TNF receptor-1, indicating that enhanced cytotoxicity is due to constructs with a luciferase reporter vector containing involucrin or transglutaminase promoters autocrine release of TNF. Accordingly, elevated TNF levels were only found upon UV plus IL- led to substantial reduction in calcium stimulated involucrin and transglutaminase promoter 1 treatment, while neither CD95L nor TRAIL with or without IL-1 affected TNF release. Since activities. Similar results were seen following treatment with a specific PLC inhibitor (U73122). activation of NF001κB protects from TRAIL- and CD95L-, but not from UV-induced apoptosis To determine the mechanism by which PLC-001γ1 was likely to alter keratinocyte differentiation, this indicates that different mechanisms are involved in UV-induced apoptosis than in CD95L- we evaluated the effect of antisense PLC-001γ1 cDNA on the rise of intracellular calcium and TRAIL-induced apoptosis. In addition, these data suggest that antiapoptotic activity of a following the calcium switch. We observed that intracellular free Ca2ϩ was reduced significantly stimulus does not only depend on its nature but also on the stimulus causing apoptosis. by this maneuver. These findings suggest that PLC-001γ1 is a critical component of the signaling pathway mediating calcium regulation of keratinocyte differentiation via its mobilization of intracellular calcium. 546 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

139 140 Telomerase Expression Affects Apoptosis in Dermal Microvascular Endothelial Cells Metalloproteinase-Deficient Mice Have Altered Contact Hypersensitivity J. Yang, D. Zhang, D. Lao, A. Cherry, C. Bangs and G. Herron M. Wang, X. Qin, R. Senior and H. Welgus Department of Dermatology and Cytogenetics, Stanford University, Stanford, California Divisions of Dermatology and Respiratory/Critical Care Medicine, Barnes-Jewish Hospital at We expressed the human telomerase reverse transcriptase (hTERT) gene in primary human dermal Washington University School of Medicine, St. Louis, Missouri microvascular endothelial cells (HDMEC) using a high efficiency, MMLV-based retroviral vector Matrix metalloproteinases (MMPs) are involved in normal physiologic processes of connective in order to determine its effect on programmed cell death. eGFP reporter gene expression showed tissue remodeling. However, excessive production of MMPs has been associated with a variety of transduction efficiencies approaching 80% in primary HDMEC using this retroviral system. chronic destructive diseases including arthritis, emphysema, skin ulcers, and abdominal aneurysms. Expression of the hTERT transgene in HDMEC was confirmed by RT-PCR using primers MMPs are expressed in T cells and macrophages, but there is a paucity of evidence for their direct within both the hTERT cDNA insert and retroviral vector. Continuously passaged hTERT(ϩ) role in immune responses. We have studied stromelysin-1 (MMP-3) and gelatinase B (MMP-9) HDMEC maintained normal epitheliod morphology, expression of FVIIIRA, CD31, telomerase deficient mice in a dinitrofluorobenzene (DNFB)-induced contact hypersensitivity (CHS) model. activity and telomere length for 60 population doublings (PD60), whereas, sham-transduced MMP-3-deficient mice showed a markedly impaired response to topical DNFB. Lymphocytes HDMEC and primary HDMEC became senescent and expressed senescence-associated 001β from the lymph nodes of DNFB-sensitized MMP-3-deficient mice failed to exhibit a proliferative galactosidase at PD30. hTERT(ϩ) HDMEC maintained normal 46, XY karyotype, exhibited response to soluble antigen dinitrobenzensulfonic acid (DNBS), but did respond identically to normal growth rates, incorporated LDL, and expressed ICAM-1 and VCAM-1 following TNF001α wild-type mice when presented with the nonspecific antigen concanavalin A. Intradermal injection stimulation similar to primary HDMEC. Apoptotic nuclear fragmentation and Apo2.7 expression of stromelysin-1 at the time of DNFB sensitization fully rescued the impaired CHS response. In in confluent hTERT(ϩ) HDMEC showed decreased ratios of apoptosis versus primary HDMEC contrast, MMP-9-deficient mice exhibited a CHS response comparable to their wildtype controls, in response to a variety of apoptotic induction agents; however, the basal level of apoptosis in but had a marked delay in resolving the reaction. Persistent inflammation was observed only with hTERT(ϩ) cells was higher than primary HDMEC at confluence. While hTERT(ϩ) cells formed DNFB sensitization and subsequent challenge, but not with the acute irritant phenol. The tubule-like structures in response to 3D collagen, differences in pattern formation were observed prolonged inflammation found in MMP-9-deficient mice with DNFB was accompanied by a lack versus primary HDMEC. We have created two additional hTERT(ϩ) HDMEC lines (currently of IL-10 production at the site of challenge. These results suggest that MMP-3 is essential for the at PD45) which behave similar to our first cell line (currently at PD70). Retroviral-mediated generation and initiation of the CHS response, while MMP-9 plays a critical role in its resolution. hTERT expression in primary HDMEC represents a novel, rapid and efficient method of creating cutaneous microvascular endothelial cell lines which maintain morphologic, phenotypic and functional properties of primary cells; however, the endothelial cell apoptotic pathway is altered by life-extension via telomerase expression.

141 142 Processing of the BP180 Ectodomain by MMP-2 in Keratinocytes Influences Extracellular Matrix Haploinsufficiency of Desmoplakin Causes Disruption of Intermediate Filament–Desmosome Organization, Cell Spreading and Generates the Linear IgA Disease Autoantigen Interactions in Palmoplantar Epidermis D. Reddy, K. McGowan, A. Russell, H. Tran, S. Herron and P. Marinkovich D. Armstrong, K. McKenna, P. Purkis,* K. Green,† R. Eady,‡ I. Leigh* and A. Hughes Stanford University, California Department of Medical Genetics, The Queen’s University of Belfast, Belfast; *Centre for Cutaneous BP180 is a transmembrane hemidesmosomal component recently shown to undergo processing Research, St Bartholomew’s and The Royal London School of Medicine and Dentistry, London; to 120 kDa. The purpose of these studies was to determine the mechanism and functional †Departments of Pathology and Dermatology, North-western University Medical School, Chicago, significance of BP180 processing. 1,10 phenanthroline and TIMP-2 inhibited BP180 processing Illinois; and ‡Department of Cell and Molecular Pathology, St John’s Institute of Dermatology, to 120 kDa in normal human keratinocyte (NHK) extracts, implicating an MMP dependent London mechanism. Although both purified MMP-9 and MMP-2 cleaved a recombinant BP180 fragment, Desmosomes are highly organised intercellular adhesive junctions that are particularly prominent only MMP-2 cleaved native BP180 extracted from NHK. TIMP-2 caused inhibition of BP180 in the epidermis. Desmoplakin, a constitutive component of the desmosomal plaque, is the most processing in NHK by IDIF microscopy using antibodies (Abs) specific to processed BP180. A abundant protein present in such junctions and plays a critical role in linking the intermediate furin inhibitor that inhibits BP180 processing was shown by gel zymography to inhibit activation filament network to the plasma membrane. Here we report the first mutation in the gene encoding of MMP-2 in NHK suggesting that the furin/MT-MMP/MMP-2 proteolytic cascade may be the desmoplakin. The identified mutation, resulting in a null allele and haploinsufficiency, was observed regulatory mechanism of BP180 processing. Both TIMP-2 and furin inhibitor reduced NHK in genomic DNA from a kindred with the dominantly inherited skin disorder, striate palmoplantar spreading. Immunomapping of MMP-2 mediated BP180 processing suggests cleavage in the amino keratoderma. Affected skin demonstrated loosening of intercellular connections, disruption of terminal NC16A domain. We produced a recombinant BP180 ectodomain whose N terminus desmosome/keratin intermediate filament interactions and a proportion of rudimentary desmosomal begins in the approximate site of MMP-2 cleavage in a mammalian expression system, and have structures. The mutation was a heterozygous C-to-T transition in exon 4 of the desmoplakin shown that this rBP180 adopts a triple helical conformation by several methods. In a survey of a gene and predicted a premature termination codon in the amino-terminal region of the peptide. number of BMZ proteins, rBP180 specifically bound to laminin-5 by solid state ligand binding This is the first inherited skin disorder in which haploinsufficiency of a structural component has assay. Abs specific to processed BP180 colocalize with laminin-5 in NHK attachment complexes been implicated and identifies dosage of desmoplakin as critical in maintaining epidermal integrity. whereas Ab to BP180 endomain shows diffuse localization in NHK suggesting that BP180’s processing may be linked to its assembly with laminin-5. Finally, rBP180, as well as processed 120 kDa BP180 molecules extracted from skin and NHK (but not full length BP180) are specifically recognized by linear IgA disease autoantibodies from the five patients we tested with active titers, implicating BP180 processing in this disease pathogenesis.

143 144 Isoform Specific Assembly of Desmosomal Cadherins into Desmosomes in Epithelial Cells The Identity and Function of CD14 LPS Receptor and Toll-like Receptor 2 in a Human S. M. Norvell, L. J. Bannon and K. J. Green Epithelial Cell Line Departments of Pathology and Dermatology, North-western University of Medical School, I. S. Song, B. Harten, L. Liu, G. P. Holley,* S. Ward,* C. A. Armstrong, H. E. Edelhauser* and Chicago, Illinois J. C. Ansel Desmosomes comprise desmoglein and desmocollin isoforms that are expressed in a cell type and Department of Dermatology, *Department of Ophthalmology, Emory University, Atlanta, Georgia differentiation specific manner. The relative contributions of these different desmosomal cadherin Lipopolysaccharide (LPS), a component of gram(–) bacterial cell wall, can induce a rapid host isoforms to desmosome assembly and are unknown. To directly compare assembly properties of inflammatory response by the activation of the innate immune system. Since the mechanism by different desmosomal cadherin isoforms, we established stable A431 cell lines ectopically expressing which LPS triggers epithelial inflammation is poorly understood, the identity and function of LPS myc-tagged desmoglein 2 (Dsg2) or myc-tagged desmocollin 2 (Dsc2), which are the endogenous binding receptors on an epithelial cell line derived from human cornea was investigated. The isoforms expressed in A431s, and compared these cell lines to A431s ectopically expressing myc- CD14 LPS receptor expression was determined by RT-PCR, Northern blot and FACS analysis. To tagged desmoglein 1 (Dsg1), the pemphigus foliaceus antigen. Dsg1 is not a normal component measure CD14 function, the intracellular calcium response to LPS and secretion of proinflammatory of A431 cells, and is only expressed in the most differentiated layers of the epidermis. Dsg1 cytokines (IL-6) and chemokines (IL-8) were determined by fura-2/AM and ELISA, respectively. localized diffusely at the cell surface and even moderate levels disrupted desmosome assembly, The expression of Toll-like receptor 2 (TLR2), the newly described putative signaling component while Dsg2 and Dsc2 colocalized with endogenous desmosomal components and desmosomes of the LPS receptor (Nature 395:284–288, 1998), was measured by RT-PCR and Northern blot appeared to assemble normally, regardless of Dsg2 or Dsc2 expression level. In cells expressing analysis. The identity of epithelial CD14 and TLR2 was confirmed by Southern blot analysis and ectopic Dsg2 or Dsc2 the levels of endogenous desmosomal cadherins were unchanged, while cell DNA sequencing. Our results demonstrate that this human epithelial cell line expresses the lines expressing Dsg1 exhibited dramatic decreases in levels of Dsc2. To address whether Dsg1- functional LPS receptor CD14 which is upregulated (2.5x) by LPS itself. LPS binding to the specific desmosome disruption was due to sequestration of plakoglobin (Pg) away from E-cadherin epithelial CD14 increases intracellular calcium and the production of IL-6 and IL-8 that can be as previously proposed, we stably introduced plakoglobin into cell lines already expressing ectopic specifically inhibited by pretreating cells with CD14 blocking antibodies. In addition we determined Dsg1, effectively increasing the available pool of plakoglobin. The resulting cell lines exhibited that these epithelial cells also express the putative LPS signaling receptor TLR2. These results 2–6 fold higher levels of plakoglobin associated with E-cadherin than control Dsg1 cell lines. indicate for the first time that a human epithelial cell is capable of expressing both functional Desmosome morphology and association with intermediate filaments appeared rescued in the CD14 LPS binding receptors as well as the TLR2 LPS signaling receptor. Understanding the majority of these lines. However, immunoblot analysis revealed that levels of Dsc2 remained low. biology of these proteins may lead to novel and specific therapeutic approaches in the management Thus, sequestration of Pg plays a critical role in Dsg1-mediated desmosome disruption but is of gram(–) bacterial infections. probably not the sole contributing factor. In summary, our results demonstrate for the first time that disruption of desmosomes by ectopically expressed desmosomal cadherins is isoform specific, and highlight the importance of tight regulation of the program of desmosomal cadherin isoform expression during epidermal differentiation. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 547

145 146 Induction of Antigen-Specific Immunosuppression with ‘‘Killer’’ Dendritic Cells Injection of naked DNA Encoding a Sequence for T Cell Receptor 001α Chain Modulates the H. Matsue, K. Matsue, M. Walters, K. Okumura,* H. Yagita,* and A. Takashima T Cell Function In Vitro and In Vivo Department of Dermatology, UT South-western, Dallas, Texas; and *Department of Immunology, G. Go¨llner, G. Mu¨ller, R. Alt, J. Knop and A. Enk Juntendo University, Tokyo, Japan Department of Dermatology, University of Mainz, Mainz, Germany Although many adhesion/costimulatory molecules facilitate physical interaction of dendritic cells Recently we could demonstrate that a highly conserved transmembrane core peptide (CP) of the (DC) with T cells, molecular recognition of MHC-associated antigenic peptides by T cell receptors T cell recptor-001α (TCR-001α) chain inhibits functional TCR assembly. In vivo, this sequence warrants antigen specificity in DC-induced T cell activation. In this study, we have engineered is important for protein–protein interactions required for the assembly of the T cell antigen DC to deliver death signals, instead of activation signals, to T cells in an antigen-specific manner. receptor. In an alloantigeneic MLR using dendritic cells as APC proliferation of CD4ϩ as well as ‘‘Killer’’ DC were created by introducing the CD95L cDNA into the fully mature DC line XS106 CD8ϩ T cells was reduced by preincubation with CP but not control peptide. No effect of CP (derived from A/J mice). A stable transfectant clone, termed ‘‘killer-XS’’, expressed abundant was observed in an anti-CD3 mAb-induced proliferation assay demonstrating the specificity of CD95L mRNA and proteins, whereas no expression was detectable in the original XS106 line or the effect for the T cell receptor assembly. We could demonstrate that CP can function as a local the clone ‘‘neo-XS’’ transfected with vector alone. The OVA-reactive CD4ϩ T cell clone MH1was immunosuppressive agent in the elicitation phase of allergic contact dermatitis. In vivo CP reduced activated maximally by OVA-pulsed neo-XS. Activation was also inducible by OVA-pulsed killer ear swelling responses by more than 50% as compared to the control peptide. We furthermore XS cells but only in the presence of anti-CD95L mAb. In standard 6 h 3H-thymidine release used plasmids containing the core sequence of the T cell recptor-001α (TCR-001α) chain. The assays, we observed that MH1 T cells were killed effectively by OVA-pulsed killer XS, but not T cell receptor transmembrane plasmids pTCRTMB and pTCRTMBA were constructed by by nonpulsed killer XS or OVA-pulsed neo-XS cells. Again, killing was blocked completely by cloning a 27-bp fragment into a secretory plasmid vector (Invitrogen, San Diego, CA). The anti-CD95L mAb. Thus, over-expression of CD95L is sufficient to create killer DC that deliver negative control plasmid pTCRTMBA contains the same 27 bp fragment in antisense orientation. death signals in an antigen specific manner. To test the in vivo funtion, we injected OVA-pulsed To assess functional activity of the DNA, HaCat cells were transfected with the plasmids by killer XS cells (4 001ϫ 105 cells/s.c. injection) into A/J mice at 6, 4, 0 d before sensitization with lipofection. CD4ϩ as well as CD8ϩ T cells were preincubated with the supernatant of transfected OVAϩ complete Freund’s adjuvant (CFA). Mice treated in this manner failed to mount footpad HaCat cells. In an alloantigeneic MLR using dendritic cells as APC proliferation of CD4ϩ or swelling response to OVA (0.5 Ϯ 0.8 001ϫ 10–2 mm; n ϭ 10), whereas marked swelling was CD8ϩ T cells was reducded by preincubation with supernatant of pTCRTMB transfected HaCat observed in mice treated with nonpulsed killer XS (8.1 Ϯ 0.8) or PBS alone (7.9 Ϯ 0.8). With cells by 70%, but not by the negative control plasmid (pTCRTMBA). Incubation with an anti- respect to antigen specificity, OVA response was abrogated by OVA-pulsed killer XS, but not CD3 mAb abolished this inhibitory effect. To assess functional activity of DNA in vivo, mice were HEL-pulsed killer XS, whereas HEL-response was abolished only by HEL-pulsed killer XS cells. sensitized with a contact allergen (1% TNCB). After three days 50 µg of plasmid DNA was To test the therapeutic efficacy, mice were first immunized with OVA ϩ CFA and received a injected into the ear skin. On day 5 a challenge dose of 0,5% TNCB was applied to the same site single s.c. injection of OVA-pulsed killer XS (4 001ϫ 105 cells) 7 d later. These mice showed no epicutaneously. PTCRTMB reduced ear swelling responses by 40% as compared to the control significant footpad swelling response to OVA (1.5 Ϯ 2.2), whereas control mice receiving plasmid. These results demonstrate that vaccination with naked DNA, coding for the T cell nonpulsed killer XS cells showed a marked response (12.7 Ϯ 1.3). We propose that CD95L- receptor exerts a local immunosuppressive effect in T cell mediated immune responses that might transduced ‘‘killer’’ DC represent an entirely new immunosuppressive protocol, which can be be useful for clinical applications in man. used clinically to selectively eliminate the pathogenic T cells that recognize a given antigen (e.g. autoantigens, allergens, or allo-antigens).

147 148 Fan, a Novel Adapter Protein of the p55 TNF-Receptor Signaling Pathway, is Involved in New Basement Membrane Functions are Dectected in an Animal Model for Junctional Epidermo- Permeability Barrier Homeostasis lysis Bullosa E. Proksch, J.-M. Jensen, D. Kreder* and M. K. S. Adam-Klages* M. C. Ryan,*† Y. Miyashita† and W. Carter† Departments of Dermatology and *Immunology, University of Kiel, Germany *Department of Medicine (Dermatology), University of Washington, Seattle, Washington; †Fred We recently showed that the p55 TNF-receptor and the downstream signaling enzymes acid and Hutchinson Cancer Research Center, Seattle, Washington neutral sphingomyelinase (A- and N-SMase) generating ceramides are important for permeability Laminin 5 is a basement membrane (BM) protein that regulates anchorage and motility of epithelial barrier function. FAN, a novel WD-repeat protein, binds to a cytoplasmatic nine amino acid cells through integrins 001α3001β1 and 001α6001β4, respectively. We used targeted disruption motif of the p55 TNF-receptor that has been shown to be both required and sufficient for the of the LAMA3 gene, which encodes the 001α3 subunit of laminin 5, to develop an animal model activation of N-SMase. We established a transgenic FAN deficient mouse model and determined for junctional epidermolysis bullosa-gravis (JEB-G). Ablation of the LAMA3 gene results in permeability barrier function as well as the capacity in skin barrier repair after tape-stripping. In neonatal lethality in homozygous null animals, concurrent with the removal of laminin 5 from addition we studied A- and N-SMase enzyme activities in these animals. In FAN deficient epithelial BM’s. Characterization of the phenotype of LAMA3 null animals has led to three compared to wild type mice we found a perturbed cutaneous permeability barrier function shown important new avenues of research. First, by directly assaying BM function, we have identified by significantly increased basal level in transepidermal water loss (TEWL) (fan–/–, 13.0 Ϯ 0.9; an alternative ligand for integrin 001α3001β1 in mutant skin. Using cryostat sections from wild fanϩ/ϩ, 9.9 Ϯ 0.6 g per h per m2,pϽ0.005, n ϭ 18) indicating a constitutive defect in barrier type and mutant animals for adhesion assays, we found that laminin 5 deficient skin could not function. After tape-stripping a significantly delayed permeability barrier repair over the entire induce stable adhesion by integrin 001α6001β4, consistent with the presence of abnormal time course monitored, at 1–24 h after barrier disruption, was noted in the FAN deficient animals. hemidesmosomes and the formation of junctional blisters. However, adhesion via integrin N-SMase enzyme activity in unstimulated FAN-deficient mice was significantly reduced (–58%, 001α3001β1 was retained in mutant animals indicating that 001α3001β1 in basal cells interacts p Ͻ 0.05, n ϭ 4); A-SMase activity was unchanged. After experimental barrier disruption we with an alternative BM ligand in laminin 5 deficient skin. Second, by analyzing the growth of found a significant increase in A- and N-SMase activities in wild type but not in FAN deficient wild type and mutant epithelial cells, we found that laminin 5 deficient-mutant cells do not mice. Reduced N-SMase activity in the epidermis of FAN deficient mice correlates well with survive in the absence of an exogenous ligand. Mutant epithelial cells could be rescued by plating impaired permeability barrier function. In summary, our in vivo results show the important role cells on exogenous laminin 5, collagen or an antibody against integrin 001α2001β1or001α6001β4. of the FAN protein coupling the p55 TNFreceptor signaling pathway to N-SMase in cutaneous These findings suggest that ligation of 001β1 or 001β4 integrins are sufficient to rescue the survival permeability barrier homeostasis. defect resulting from ablation of laminin 5. Third, by examining developing incisors from normal and mutant animals, we have established a regulatory role for laminin 5 in tissue development. Analysis of developing incisors revealed abnormalities in late stage differentiation of the enamel organ in mutant animals, providing a biological basis for the enamel hypoplasia that is detected in JEB-G patients. Thus, using an animal model for JEB-G, we have been able to detect a new BM ligand in skin and show that events downstream of adhesion are regulated by laminin 5.

149 150 Targeted Disruption of the Type VII Collagen Gene in Mice Results in Severe Blistering Ultraviolet Light Induced Apoptosis is Mediated by Nuclear as well as by Membrane Effects Phenotype: A Model for Recessive Dystrophic Epidermolysis Bullosa D. Kulms, B. Po¨ppelmann, D. Yarosh, T. Luger, J. Krutmann and T. Schwarz M. Ma¨nnikko¨, S. Heinonen, D. Whitaker-Menezes, G. Murphy, J. Klement and J. Uitto Department of Dermatol., University of Mu¨nster, Mu¨nster, Germany; AGI-Dermatics, Freeport, Departments of Dermatology and Cutaneous Biology, and Pathology, Thomas Jefferson University, New York; Department of Dermatol., University of Du¨sseldorf, Du¨sseldorf, Germany Philadelphia, Pennsylvania Ultraviolet light (UV) induced DNA damage is a crucial event in UV-mediated apoptosis. On The dystrophic forms of epidermolysis bullosa (DEB) are caused by mutations in the gene (Col7a1) the other hand, direct activation of the death receptor CD95 by UV was recently shown, implying encoding type VII collagen, the major component of anchoring fibrils. To generate an appropriate that UV-induced apoptosis can be initiated at the cell membrane through death receptor clustering. animal model of DEB, we have developed a type VII collagen deficient mouse by targeted This study was performed to measure the relative contribution of nuclear and membrane effects homologous recombination. The targeting vector resulted in a replacement of 5.5 kb of the gene, in UV-induced apoptosis. UV-mediated DNA damage can be reduced by treating cells with corresponding to exons 46–69, by neo gene in reverse orientation. This resulted in elimination of liposomes containing the repair enzyme photolyase. Addition of photolyase after UV exposure of most of the amino-terminal half of the triple-helical domain of type VII collagen. Col7a1 ϩ/– HeLa cells reduced the apoptosis rate significantly, while empty liposomes had no effect. UV mice heterozygous for the deletion were phenotypically normal. Screening of offspring from exposure at 4°C which prevents CD95 clustering also reduced the apoptosis rate but to a much Col7a1 ϩ/– parents by Southern analysis and PCR revealed that Col7a1 –/– (null) mice were lesser extent. Osmotic shock does not induce DNA damage but was recently shown to induce born with extensive cutaneous blistering, and they also developed erosions in the feet. Oral blisters surface receptor clustering. Accordingly, exposure of HeLa cells to 1 M sorbitol induced apoptosis. also developed within a few days of life. Complications of the blistering resulted in demise of In contrast to UV, osmotic apoptosis was almost completely diminished at 4°C. Cells transfected these animals usually during the first week of life. Histopathology revealed subepidermal blistering, with a dominant negative mutant of FADD, a signaling protein of the CD95 pathway, were which by transmission electron microscopy was shown to be below the lamina densa. Electron resistant to osmosis, indicating that osmotic apoptosis is exclusively initiated at the membrane. microscopy also revealed the absence of anchoring fibrils. Immunohistochemical staining with a When cells were exposed to UV at 4°C and treated with photolyase, UV-induced apoptosis was rabbit polyclonal antibody raised against recombinant NC1 domain of human type VII collagen, also completely prevented. These data indicate that in UV-mediated apoptosis nuclear effects are which recognizes type VII collagen in normal mice in a linear pattern at the dermal-epidermal predominant in comparison to membrane events. Nevertheless, for the complete apoptotic response junction, was negative in the skin of Col7a1 –/– mice. Thus, these mice recapitulate the clinical both are necessary. Thus, this study shows that nuclear and membrane effects are not mutually features, as well as immunohistochemical and ultrastructural findings, in recessive dystrophic EB exclusive and that both are essential for a complete UV response. in humans. These mice provide an excellent model to study the pathomechanisms of DEB and serve as a system to test therapeutic approaches, including gene replacement, towards the cure of this devastating skin disease. 548 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

151 152 Elucidation of Apoptotic Signaling Pathways Following Activation of the 75 kDa Neurotrophin The Human Nude Phenotype: Congenital Alopecia and Severe T-Cell Immunodeficiency Receptor Associated with a Nonsense Mutation in the Whn Gene S. Zhai, M. Yaar, W. Reenstra and B. A. Gilchrest J. Frank,* C. Pignata,‡ A. A. Panteleyev,* D. M. Prowse,§ H. Baden,§ L. Weiner,§ L. Gaetaniello,‡ Boston University School of Medicine, Boston, Massachusetts W. Ahmad,* N. Pozzi,‡ P. B. Cserhalmi-Friedman,* D. Gordon,ϩ J. Ott,ϩ J. L. Brissette§ and The 75 kDa neurotrophin receptor (p75) is strongly expressed in keratinocytes, melanocytes and A. M. Christiano*† neurons and has been implicated in apoptosis of these cells under certain conditions. When Departments of *Dermatology and †Genetics and Development, Columbia University, New York, neurotrophins activate p75 together with receptors of the Trk family, p75 evokes a survival signal. New York; ‡Department of Pediatrics, ‘‘Federico II’’ University, Naples, Italy; §Cutaneous Biology However, when p75 is activated alone, it may signal for apoptosis by stimulating within minutes Research Center, Massachusetts General Hospital, and Department of Dermatology, Harvard sphingomyelin turnover and ceramide generation. Still, the sequence of events linking p75 Medical School, Charlestown, Massachusetts; ϩLaboratory of Statistical Genetics, The Rockefeller stimulation to ceramide generation and apoptosis remain largely unknown. To investigate p75 University, New York, New York early signaling, NIH-3T3 cells engineered to constitutively express human p75 (3T3-p75), were The nude mouse phenotype is characterized by congenital absence of hair and severe immuno- β β stimulated with a known p75 ligand 001 amyloid (001 A), and the distribution of p75 on the deficiency, resulting from mutations in the whn gene (winged-helix-nude), which encodes a cell surface was analysed using immunohistochemistry and confocal laser microscopy. Within β forkhead/winged helix transcription factor family member with restricted expression in thymus minutes 001 A-treated cultures displayed aggregation of p75, while the baseline, homogeneous and skin. Identification of the human counterpart of the nude mutation has remained elusive, cell surface distribution of p75 did not change in diluent treated cultures. Furthermore, 3T3-p75 β presumably because affected individuals succumb to the immunodeficiency before the complete stimulated with 001 A in the presence of a bifunctional crosslinker and then reacted with anti absence of hair can be appreciated. Recently, the simultaneous occurrence of severe functional T- p75 antibodies displayed on western blots in addition to the expected 75 kDa band also a ~220– cell immunodeficiency, congenital alopecia and nail dystrophy (MIM 601705) in two female 230 kDa band, consistent with receptor trimerization, as reported for other apoptotic signaling pathways. Moreover, similar to signaling initiated by the apoptotic TNF-001α and Fas receptors, siblings from a consanguineous Italian family was reported. One sibling survived due to a bone 001βA activation of p75 strongly induced the transcription of the immediate early c-jun mRNA, marrow transplantation which corrected the immunodeficiency, but not the congenital alopecia. We sought to test the hypothesis that this syndrome represented a candidate disorder for underlying stimulated the stress-activated c-Jun NH2-terminal kinase (JNK) as measured by phosphorylation of its substrate [GST-cJun (1–79)], activated caspase-3 to cleave its substrate [poly (ADP mutations in the human whn gene. We found suggestive evidence of linkage to the whn locus on ribose)polymerase], and induced the characteristic DNA fragmentation into multimers as measured human chromosome 17 (Zmax ϭ 1.32), identified a homozygous nonsense mutation in affected by TUNEL analysis and DNA ladder formation. To determine if the initial step of p75 aggregation individuals, and localized the expression of human whn to tissues involved in pathogenesis. These is required for initiation of apoptosis, 3T3-p75 were pretreated with an HPLC purified cyclic findings implicate a forkhead/winged helix family member in the etiology of such diverse peptide (CATDIKGAEC) that binds the ligand binding site of p75, and then cultures were developmental defects as congenital absence of the hair and athymia in humans. stimulated with 001βA or with diluent alone. The cyclic peptide inhibited p75 aggregation, decreased c-jun mRNA induction, reduced GST-cJun (1–79) phosphorylation, and suppressed cellular apoptosis. The universality of the pathway was confirmed by treating UV-irradiated keratinocytes (50 mJ per cm2, metered at 285 Ϯ 5) with the cyclic peptide. Cyclic peptide blocking of p75 decreased c-jun transcription that was otherwise prominent in UV-irradiated diluent-treated keratinocytes. Our data identify for the first time the initial signaling events that follow p75 activation and suggest that signaling through p75 requires receptor aggregation. Hence, p75 mediated apoptosis could be abrogated by cyclic peptides that isolate the receptor, preventing its activation.

153 154 Agouti Signaling Protein Inhibits Melanogenesis Primarily by Binding to the Melanocortin- Glucocorticoids Induce a Near-Total Suppression of Hyaluronan Synthase mRNA in Dermal 1 Receptor Fibroblasts and in Osteoblasts: A Molecular Mechanism Contributing to Organ Atrophy Z. Abdel-Malek, M. Furumura,* L. Lamoreux,† M. Ollmann,‡ G. Barsh‡ and V. Hearing* W. Zhang, C. Watson, C. Liu, K. Williams and V. Werth Dept of Dermatology, Univ of Cincinnati, Cincinnati, Ohio; *Laboratory of Cell Biology, NCI, Department of Dermatology, University of Pennsylvania, Philadelphia, Pennsylvania; Department NIH, Bethesda, Maryland; †Department of Veterinary Pathology, Texas A&M University, College of Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania Station, Texas; ‡Howard Hughes Medical Institute, Stanford University, Stanford, California Topical and systemic glucocorticoids induce an atrophy of skin, bone, and other organs that is Agouti signaling protein (ASP) is known to antagonize the melanogenic effects of 001α-melanocyte characterized by decreased tissue content of glycosaminoglycans, in particular hyaluronic acid stimulating hormone (001α-MSH) on mouse follicular melanocytes, resulting in the switch from (HA). We took advantage of the recent cloning of the three mammalian hyaluronan synthase eumelanin to pheomelanin synthesis. We have shown that ASP completely abrogates the mitogenic (HAS) enzymes, HAS-1, Ϫ2, and Ϫ3, to explore the molecular basis of this side-effect. Northern and melanogenic effects of 001α-MSH on normal human melanocytes, and competes with 001α- blots performed on RNA extracted from cultured dermal fibroblasts and the MG-63 osteoblast- MSH for binding to the melanocortin 1 receptor (MC1R). Since 001α-MSH stimulates like osteosarcoma cell line indicated that HAS-2 is the predominant HAS mRNA in these cells. eumelanogenesis in normal human melanocytes, it is expected that ASP would induce pheomelanin Incubation for 24 h with 1 µM dexamethosone suppressed HAS-2 mRNA to ~2.0% of control synthesis. We have investigated whether ASP functions primarily by binding to MC1R, or values in fibroblasts and to ~17% in the osteoblast line. A time course performed in cultured additionally binds to another yet-unidentified receptor. For that, we compared the responses of dermal fibroblasts indicated suppression of HAS-2 mRNA to 28% of control by 1 h and to 1.2% cultured congenic C57BL/6 J-Eϩ/Eϩ (B6) mouse melanocytes that express wildtype MC1R, of control by 2 h after addition of dexamethasone. Nuclear run-off experiments showed a 3-fold C57BL/6 J-e/e (e/e) melanocytes that express a mutated MC1R that is uncoupled to adenylate suppression of HAS-2 gene transcription in nuclei from dexamethasone-treated fibroblasts, which cyclase, and C57BL/6 J-Eso/Eso (Eso/Eso) melanocytes that express a mutated MC1R that is is unlikely to fully explain the 50–80-fold reduction in message levels. Experiments with constitutively active in the absence of ligand binding, to ASP. We found that ASP markedly actinomycin-D demonstrated a proportionately larger effect on message half-life, which was 3 h reduced basal tyrosinase activity of B6 melanocytes. In addition, ASP inhibited the melanogenic in the absence of glucocorticoids but less than 11 min after treatment with dexamethasone. In effect of 001α-MSH on these cells by significantly reducing the 001α-MSH induced cAMP level, conclusion, glucocorticoids induce a rapid, near-total suppression of HAS-2 message levels, largely tyrosinase activity, and protein levels of tyrosinase, TPR-1, and TRP-2. However, neither 001α- mediated through changes in message stability, but also involving reduced gene transcription. We MSH nor ASP altered tyrosinase activity or the protein levels of tyrosinase, TRP-1 or TRP-2 in infer that these effects account for the decrease in HA seen in glucocorticoid-treated skin, bone, e/e or Eso/Eso melanocytes. These findings indicate lack of responsiveness of e/e or Eso/Eso to and other organs. 001α-MSH or ASP, and clearly demonstrate that ASP elicits its effects mainly by binding to the MC1R. Our results underscore the significance of normal MC1R expression in the modulation of melanogenesis in mammalian melanocytes by 001α-MSH or ASP.

155 156 Tyrosinase Expression is Regulated by p53 The Neuropeptide Substance P Activates NFAT- and NFkhB-Dependent Adhesion Molecule M. Khlgatian, P. Asawanonda, M. Eller, M. Yaar, M. Fujita,* D. A. Norris* and B. A. Gilchrest Gene Expression in Microvascular Endothelial Cells Boston University School of Medicine, Boston Massachusetts and *University of Colorado School K. Quinlan, S. Naik, C. Armstrong, J. Ansel and S. Caughman of Medicine, Denver, Colorado Emory University School of Medicine, Atlanta, Georgia Tyrosinase is the rate limiting enzyme for melanin synthesis. In cultured melanocytic cells its Upon stimulation, cutaneous sensory nerves release neuropeptides such as substance P (SP), which expression is induced by DNA damage after ultraviolet (UV)- or X-irradiation or treatment with modulate responses in the skin by activating a number of target cells via neurokinin receptors other DNA damaging agents. Small DNA fragments, particularly thymidine dinucleotide (pTpT), (NK-R). We have previously demonstrated that SP preferentially binds to the NK-1R on human also induce tyrosinase mRNA and increase melanogenesis when added to cultured melanocytic dermal microvascular cells (HDMEC), resulting in increased intracellular Ca2ϩ and induction of cells or applied to intact skin. DNA damage is known to activate p53, and we have shown that intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) pTpT also activates p53. Together these data suggest that p53 may regulate tyrosinase gene expression. In the current studies, we identify specific elements in the regulatory regions of expression. We therefore examined tyrosinase mRNA level in a melanoma line derived from an ICAM-1 and VCAM-1 genes as necessary and sufficient for SP-dependent transcriptional activation. early primary tumor (WM35) and in 2 lines derived from WM35 engineered by transfection to DN SP treatment of HDMEC leads to activation and binding of the transcription factor NFAT to – constitutively express a dominant negative p53 mutant (WM35-p53 ) lacking p53 transcriptional 191/–170 region of the ICAM-1 gene, and NFknB (p65/p50) to tandem NFkFB binding sites activity, or containing the plasmid vector alone (WM35-pCMV) as a control. As expected, p53 at –76/–52 of the VCAM-1 gene. The SP-elicited intracellular Ca2ϩ signal was required for protein levels were far higher in WM35-p53DN than in WM35 and WM35-pCMV. Interestingly, activation and subsequent binding of both NFAT and NFkiB. TNFa. stimulation of HDMEC, tyrosinase mRNA was considerably lower in these cells expressing the high levels of the dominant which also induces ICAM-1 and VCAM-1 gene transcription via NFkBB-dependent pathways, negative p53 than in cells expressing wild type p53, suggesting that p53 protein, even inactive, 2ϩ down-regulates tyrosinase mRNA. To determine the effect of p53 activity on tyrosinase mRNA, does not involve either Ca mobilization or activation and binding of NFAT. The transacting cultures were sham-or UV-irradiated (10 mJ per cm2, metered at 285 Ϯ 5 nm) or were treated factor induction by SP was specific, since a selective NK-1R antagonist blocked SP-activation with 100 µM pTpT, both conditions known to activate p53. Total cellular RNA was isolated and subsequent ICAM-1 and VCAM-1 DNA binding by NFAT and NFknB, respectively. These data not only demonstrate a novel coincident activation of NFAT and NFkFB via SP-induced after stimulation and the tyrosinase mRNA level was determined by northern blotting. In UV- 2ϩ irradiated WM35 and WM35-pCMV cells, p53 protein levels increased within 1–3 h and there intracellular Ca mobilization, they also indicate a crucial role for neuropeptides in modulating was a concomitant initial down-regulation of tyrosinase expression, consistent with p53 protein localized cutaneous inflammatory responses. downregulating tyrosinase mRNA. By 48 h there was a ~3-fold upregulation of tyrosinase mRNA. In contrast, in pTpT-treated WM35 and WM35-pCMV cells, in which p53 was activated within 6 h without changing the protein level, there was a 2–3-fold up-regulation of tyrosinase mRNA within 24 h that persisted through 48 h. No upregulation of tyrosinase mRNA was observed in WM35-p53DN after UV-irradiation or pTpT simulation. The data suggest that p53, regardless of activation status negatively regulates tyrosinase transcription, while activated p53 increases tyrosinase mRNA level through enhanced gene transcription. This would be consistent with the known ability of p53 to sequester TATA-box-binding proteins for which the tyrosinase promotor contains binding sequences, and of activated p53 to transcriptionally upregulate genes involved in protective responses to UV irradiation. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 549

157 158 Aberrant Regulation of TNFa in the Skin of p53 Knockout Mice Before and After UV-B Irradiation Regulation of Human Involucrin Promoter Activity via a Specific MAPK Signal Transduction J. Kibitel, C. Anderson, C. Elmets, D. Brash and D. Yarosh Cascade Applied Genetics Inc. Dermatics, Freeport, New York; Department of Dermatology, Case Western T. Efirmova, P. T. LaCelle, J. T. Welter and R. L. Eckert Rerserve University, Cleveland, Ohio;. Department of Radiation Therapy, Yale University, New Physiology and Biophysics, Dermatology, Biochemistry and Oncology, Case Western Rerserve Haven, Connecticut University School of Medicine, Cleveland, Ohio TNF001α is a primary cytokine responsible for inflammatory and immunosuppressive responses Limited knowledge is available regarding the signal transduction cascades that regulate gene in skin following UV. In order to better understand its genetic regulation, the induction of expression during keratinocyte differentiation. In the present study, we investigate the cascade that TNF001α in p53 and c-fos knockout mouse skin after UV-B irradiation was observed by mediates the increase in involucrin mRNA levels when keratinocytes are treated with the fluorescence immunohistochemistry. In wild type mice, TNF001α increased immediately after keratinocyte differentiating agent, 12Ϫ0-tetradecanoyl phorbol-13-acetate (TPA). PKC and irradiation, declined at 6 h, and then rose again at 12 h before failing by 24 h. This pattern was tyrosine kinase inhibitors block the TPA-dependent increase in hINV mRNA level and promoter confirmed by RT-PCR of TNF001α mRNA message in cultured epidermal cells. It was not due activity. We show that the relevant response element is located within the promoter proximal to an increase in TNF001α receptors, since TNFR-I actually declined after UV-B exposure. regulatory region (PRR) and includes an AP1 site, AP1Ϫ1. Cotransfection of the hINV promoter Induction was also found in c-fos gene knockout mice deficient in the AP-1 transcription factor, with dominantnegative forms of Ras, MEKK1, MEK1, MEK7, MEK3, p38/RIK and c-Jun demonstrating that, although AP-1 signaling is required for some UV responses, it is not required inhibit the TPA-dependent increase. Wild type MEKK1 enhances promoter activity and the for TNF001α activation. However, in homozygous p53 knockout mice the basal level of activity can be inhibited by dominant-negative MEKK1, MEK1, MEK7, MEK3, p38/RIK and TNF001α in the epidermis was greatly elevated without UV irradiation. This level declined and cJun. In contrast, wild type Raf-1, ERK1, ERK2, MEK4, or JNK1 produced no change in remained constant following irradiation. This implies that p53 signaling directly or indirectly activity and the dominant-negative forms of these kinases failed to suppress TPA-dependent represses TNF001α gene expression. Overexpression of the immunosuppressive cytokine TNF001α transcription. Treatment with an S6 Kinase (S6K) inhibitor, or transfection with constitutively may contribute to the carcinogen-susceptibility of p53 defective skin. active S6K produced relatively minor changes in promoter activity, ruling out a regulatory role for S6K. These results suggest that activation of involucrin transcription involves a pathway that includes PKC, Ras, MEKK1, MEK3, and p38/RK. Additional pathways that transfer MEKK1 activation via MEK1 and MEK7 also function, but the downstream targets of these kinases need to be identified. AP1 transcription factors appear to be the ultimate target of this regulation.

159 160 Functional Analysis of Human LAMA3A Promoter Reveals Cell Type-Specificity Through AP- Nuclear Factor (NF) kB Abnormalities in Lymphocytes From Patients With Systemic Lupus Eryth- 1 Binding Sites ematosus T. Virolle, M. Monthouel, J. Ortonne and D. Aberdam H. K. Wong, G. Dennis,* G. M. Karnmer† and G. C. Tsokos INSERM U385, Nice, France. Department of Clinical Physiology, Walter Reed Army Institute of Research, Washington, D.C.; Laminin-5, an adhesion ligand of specialized epithelia presents a complex transcriptional regulation *Walter Reed Army Medical Center, Washington, D.C.; †Bowman Gray School of Medicine, under both physiological and pathological conditions. We have cloned the 5’-flanking region of Winston-Salem, North Carolina the gene coding for the human 001α3A chain (LAMA3A) and analyzed its promoter activity. Immune cell abnormalities are prominent in lupus patients. In T cells, these abnormalities include Transient expression of a luciferase reporter gene under the control of serially deleted 5’-flanking abnormal (increased or decreased) production of various lymphokines, such as IL-2, aberrant T sequences revealed that the promoter contains both positive and negative domains. Furthermore, cell receptor-initiated calcium responses, protein tyrosine phosphorylation and deficient CD3 z the proximal promoter which contains three AP-1 sites was sufficient for keratinocyte cell type- chain expression. These previous findings support the presence of underlying biochemical defects specific expression. A single copy of this 204 bp-genomic fragment was able to confer high in lupus T lymphocytes. Because NFkB presumptively contributes to the transcription of numerous keratinocyte-specific expression to TK heterologous promoter. Simultaneous mutations of the inflammatory genes prominent in rheumatic diseases and has been shown to be a molecular target three AP-1 sites cooperatively abated promoter activity and keratinocyte-specific stimulation of of anti-inflammatory drugs, we sought to characterize the functional role of the NFkB protein the TK heterologous promoter. Removal of the sequences located between the AP-1 sites does complex in lupus lymphocytes. Freshly isolated T lymphocytes from lupus patients (n ϭ 22), not alter significantly the cell-specific activity of the promoter, although the distance between the patients with other systemic rheumatic diseases (n ϭ 7), and normal individuals (n ϭ 17) were AP-1 sites seems critical for optimal transactivation in keratinocytes. Electrophoretic mobility shift activated physiologically via the T cell receptor with anti-CD3 and anti-CD28 antibodies to assays showed that each LAMA3A AP-1 site formed a single protein-DNA complex containing assesses proximal membrane signaling and with phorbol myristate acetate (PMA) and calcium JunD and JunB in keratinocytes while in fibroblasts predominantly JunD was bound. These results ionophore to bypass membrane-mediated signaling events. We measured the NFkB binding provide evidences that the AP-1 sites in the LAMA3A promoter play a crucial and indispensable activity in nuclear extracts by gel shifts analysis. When compared to normal cells, the activation role in keratinocyte-specific expression of the gene. The small difference in the individual AP-1 of NFkB in lupus cells was decreased greater than 1 SD in 90%, and 2 SD in 54%. NFkB binding components bound in keratinocytes and fibroblasts point to subtle cell-type specific differences in activity was undetectible in several patients who were not receiving any medications, including LAMA3A gene regulation. However, by western blot analysis, JunB protein level is identical in corticosterolds. The NFkB activity in lupus lymphocytes did not change to normal levels in the two cell types and do not show any difference in overall phosphorylation state of JunB. We follow-up studies. In supershift experiments using specific antibodies, lymphocytes from patients have further shown that oxidation-reduction modification of AP-1 could not explain the difference who displayed defective NFkB activity did not have p65/p50 heterodimers. Western analysis in cell-specific transactivation and Southern blot analysis have excluded that methylation state of demonstrated reduced p65 protein in these lupus lymphocytes. As the NFkB heterodimer is genomic DNA can be responsible for that cell specificity. Therefore, these results indicate that transcriptionally active in comparison to the p50 homodimer, this novel finding may provide the keratinocyte-type specificity of the AP-1 elements in the LAMA3A promoter context is not insight on the origin of abnormal cytokine gene transcription in lupus lymphocyte determined only by the composition of the AP-1 complexes but suggest the presence of an epithelial-type specific nuclear protein which might interact with the AP-1 complexes only when all three-dimensional requirements involving contacts wit the DNA and the AP-1 proteins are satisfied. The identification of such an epithelial-specific cofactor should further our understanding of the molecular mechanisms regulating LAMA3A gene and more generally epithelial gene expression.

161 162 Regulation of Dendritic Cell-Specific Expression of the Dectin-2 gene CCAAT/Enhancer Binding Proteins – Required Regulators of Human Involucrin Promoter M. Bonkobara, P. Zukas, S. Shikano and K. Ariizumi Activity Department of Dermatology, University of Texas South-western Medical Center, Dallas, Texas C. Agarwal, T. Efimova, J. Welter, J. Crish and R. Eckert We identified the dectin-2 (Dec2) gene, the expression of which is restricted to cells of dendritic Physiology and Biophysics, Dermatology, Biochemistry and Oncology, Case Western Reserve cell (DC) lineage. We have also reported that Dec2 expression is regulated by a novel transcription University, Cleveland, Ohio unit (5’-flanking region of 3.2 kb) that includes enhancer (900 bp), repressor (1300 bp) and 12Ϫ0-tetradecanoyl phorbol-13-acetate (TPA), is a potent inducer of keratinocyte differentiation promoter (123 bp) regions. To determine whether promoter or enhancer controls DC specificity, and of involucrin gene expression. In the present study we show that a C/EBP site in the hINV we constructed luciferase vectors in which the Dec2 promoter or enhancer was replaced with a promoter proximal regulatory region (PRR) is required for promoter activity. Mutation of the nonspecific E1b promoter or SV40 enhancer. XS-DC or NS-fibroblast lines were transfected with C/EBP site results in the loss of basal and TPA-responsive activity. Gel mobility supershift analysis the vectors, and luciferase activities were assayed. Although the maximum amount of DC-specific shows that C/EBP001α binding to this site is increased by TPA treatment, but immunoblot expression was seen when Dec2 promoter and Dec2 enhancer were both present, DC-specific analysis shows that C/EBP001α protein levels are not increased. Transfection of C/EBP001α expression was retained (albeit to a lesser degree) when Dec2 promoter was coupled to SV40 increases hINV promoter activity to an extent comparable to TPA treatment. Mutation of the C/ enhancer or when Dec2 enhancer was coupled to E1b promoter. Focusing on regulation of the EBP binding site eliminates these responses. Transfection experiments using GADD153, a promoter region, we found that deletion of an 88-bp sequence (nt –123 to –36) abrogated dominantnegative C/EBP factor, to create C/EBP-null conditions, confirm that C/EBP factors transcriptional activity in XS-DC. Moreover, gel mobility shift assays revealed a 30-bp sequence are absolutely required for promoter activity and TPA responsiveness. Dose–response transfection (nt –94 to –65), which does not contain known nuclear factor-binding sites, but can form four experiments show that C/EBP001β and C/EBP001δ inhibit both TPA- and C/EBP001α- different complexes with nuclear proteins prepared from XS-DC. Two of these complexes were dependent promoter activation, indicating functional differences among C/EBP family members. nonspecific since they were detectable in several non-DC lines, whereas the other two were Additional studies suggest that C/EBP factors collaborate with AP1 factors at closely spaced sites detected highly in XS-DC but only minimally in macrophages. We conclude that promoter and within the hINV PRR. These results show that C/EBP transcription factor activity is, necessary enhancer, separately and additively, regulate DC-specific expression of the Dec2 gene. An 88-bp for basal and TPA-response hINV promoter activity, and suggest that C/EBP001α, which is sequence (nt –123 to –36) is required for promoter activity. Finally, we have identified four highly expressed in the suprabasal epidermal layers, may have a role in driving differentiation- nuclear proteins capable of binding to a 30-bp site within this required sequence. These novel specific hINV gene expression. protein–DNA interactions may be relevant to the DC specificity of Dec2 gene expression. 550 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

163 164 The Book of Opposites: Molecular Mechanisms of Glucocorticoid Action in Epidermis PPAR001α-Activation Promotes Epidermal Keratinocyte Differentiation In Vivo N. Radoja, S. Jho, M. Im, I. Freedberg and M. Tomic-Canic L. Ko¨mu¨ves, K. Hanley, A-M. Lefebvre, M-Q. Man, M. Williams, P. Elias, J. Auwerx and The Ronald O. Perelman Department of Dermatology, NYU Medical School, New York, K. Feingold New York Departments of Dermatology, Medicine, and Pediatrics, University of California San Francisco; Glucocorticoids (GC) are important regulators of epidermal growth, differentiation and homeostasis, and Veterans Affairs Medical Center, San Francisco, California; and U325 INSERM, Institute and are extensively used in treatment of skin diseases. Utilizing keratin gene expression as a Pasteur, Lille, France paradigm of epidermal physiology and pathology, we have developed a model system to study We have recently shown that PPAR activators (clofibrate or Wy14,643) stimulate differentiation molecular mechanism of GC action in skin. We found that GC have a more specific regulatory in cultured human keratinocytes and accelerate epidermal development and permeability barrier pattern than retinoic acid (RA) or thyroid hormone (T3). Specifically, GC repress the expression formation in fetal rat skin explants. The aim of the present study was to determine the role of of the basal cell-specific keratins K5 and K14 and disease-associated keratins K6, K16, and K17, PPAR activation in adult epidermis. Topical treatment of several strains of adult mice with PPAR but not the differentiation specific keratins K3 and K10 or the simple epithelia specific keratins activators resulted in decreased epidermal thickness, coupled with increased expression of involucrin, K8, K18, and K19. We have identified the negative recognition elements (nGREs) in all five profilaggrin/filaggrin, and loricrin (detected by in situ hybridization and immunohistochemistry). regulated keratin gene promoters. The function of the keratin nGREs is instructive, i.e. they Moreover, topically applied PPAR activators increased apoptosis (as measured by TUNEL assay) interact with the receptor (GR) in a specific way that conveys negative regulation. Detailed while decreasing cell proliferation (detected by BrdU incorporation). Topical treatment with footprinting revealed that GR binds to keratin nGREs as four monomers. Using cotransfection PPAR activators did not disturb baseline epidermal permeability barrier, but resulted in an and antisense technology, we have found that coregulators NCoA and HAT are part of the GR accelerated recovery of barrier function following acute barrier abrogation. To determine whether complex that leads to suppression of keratin gene transcription. In addition, GR blocks induction these effects were due to specific PPAR001α activation, we analyzed keratinocyte gene expression in of keratin gene expression by AP1. We conclude that GR suppresses keratin gene expression PPAR001α knockout mice which display focal parakeratosis, suggestive of impaired differentiation. through two independent mechanisms: first directly, through interactions of keratin nGREs, four Compared with the wild-type epidermis, involucrin, profilaggrin/filaggrin, and loricrin expression GR monomers, NCoA and HAT and second indirectly, by blocking the induction by AP1. (detected by in situ hybridization and immunohistochemistry) were decreased in PPAR001α knockout mice. Moreover, topical clofibrate treatment did not increase epidermal differentiation in PPAR001α knockout mice. This study shows that PPAR001α activation promotes keratinocyte gene expression, enhancing epidermal differentiation and inhibiting proliferation in intact adult skin. Moreover, endogenous activation of PPAR001α may play a role in the regulation of normal keratinocyte differentiation.

165 166 A Minimal 120 bp Promoter is Sufficient for Sustained, Tissue-Specific Expression of Keratin k6a Sp1 as a Regulator of Tissue-Specific Expression of Human Involucrin – Sp1 Dependent Activation in Transgenic Mice of Expression in Nonepithelial Cell Types D. Mahony, S. Karunaratne and J. A. Rothnagel E. Banks, J. Crish and R. Eckert Department of Biochemistry, University of Queensland, Brisbane, Australia Physiology and Biophysics, Dermatology, Biochemistry and Oncology, Case Western Reserve The analysis of K6 expression is complicated by the presence of multiple isoforms that are University, Cleveland, Ohio expressed constitutively in a number of internal stratified epithelia, in palmoplantar epidermis and Human involucrin (hINV) gene is selectively expressed in stratifying epithelial cells lining external the companion and outer root sheath layers of the hair follicle. In addition, K6 expression can be body surfaces. Previously, we characterized the hINV, promoter 5Ј distal regulatory region (DRR) induced in interfollicular epidermis by wounding stimuli, phorbol esters or retinoic acid. In order located between nucleotides –2473/2088 upstream of the transcription start site. This region is to define the critical cis-elements involved in the regulation of mouse K6a (the dominant isoform required for optimal hINV gene expression. The DRR contains weak and strong activator in mice), we generated transgenic animals with two different promoter constructs linked to the elements. The strong activator is comprised of AP1 and SP1 binding sites that combine to drive E. coli 001β-galactosidase reporter gene. These constructs contained either 1.3 kb or 0.12 kb of high level promoter expression in human keratinocytes. In the present study, we show that the 5Ј flanking sequences, 0.8 kb of 3Ј flanking sequences, the 5Ј and 3Ј untranslated regions and the hINV promoter is expressed in a cell-specific manner in vitro and that the DRR contains elements first intron of the mouse K6a gene. Microinjection and subsequent implantation of embryos that are partially responsible for cell-type specific expression of hINV. hINV promoter activity is resulted in 25 viable pups carrying the 1.3 kb promoter construct and 33 pups with the 0.12 kb barely detectable in 3T3 fibroblasts or HEK-293 human embroynic kidney cells. Reporter plasmids promoter construct. Several of the strongest expressors (determined from ear biopsies) were chosen containing the full-length promoter or the isolated DRR can, however, be activated in 3T3 and to establish transgenic lines and their progeny were used for analysis of transgene expression. HEK-293 cells by cotransfection with a plasmid encoding the transcription factor Sp1. Consistent Ectopic expression was not observed for either construct as determined by RT-PCR analysis of wih the lower hINV promoter activity, immunoblot studies indicate that Sp1 protein levels are spleen, liver, brain, heart, kidney and bladder mRNAs. Double-label immunofluorescence analysis lower in 3T3 and HEK293 cells compared to human epidermal keratinocytes. Increased Sp1 demonstrated coexpression of both K6a constructs with endogenous K6 in the hair follicle, tongue protein in. transfected 3T3, and HEK-293 cells correlates with increased promoter activity: In epithelium, footpad epidermis and nail bed indicating that these constructs retained keratinocyte- addition, Sp1 transfection activates expression of the endogenous hINV gene in HEK-293 cells. specific expression with no discernable difference in the expression between the two transgenes. These studies suggest that Sp1 may play a role in cellspecific expression of hINV. Moreover, expression of both constructs was induced in interfollicular epidermis by the application of retinoic acid or phorbol 12-myristate 13-acetate. In addition, we observed expression of both transgenes and endogenous K6 in the interfollicular epidermis of snout tissue. Quantitative chemiluminescent assays of 001β-galactosidase activity in protein extracts prepared from tongue, snout and back skin verified that there was no difference in expression levels between the 1.3 and 0.12 kb constructs. Therefore, this study has revealed that a minimal K6a promoter of 120 bp can direct strong, sustained and tissue specific expression and indicates that the critical cis-elements for K6a expression lie within this sequence.

167 168 Stearoyl CoA Desaturase (SCD1) Gene is Expressed in Mouse Pilosebaceous Apparatus and is Functional Defects of Cx26 Due to Mutations in Two Families with Dominant Palmoplantar defective in the Asebia Mutant Mouse Keratoderma and Deafness Y. Zheng, K. J. Eilertsen, L. Ge, S. Prouty, G. P. Sreekumar, J. Sundberg,* K. Stenn and S. Parimoo S. Bale, T. White, C. Munro, A. Taylor and G. Richard Skin Biology Research Center, Johnson & Johnson Consumer Companies, Skillman, New Jersey; Genetic Studies Section, NIAMS, NIH, Bethesda, Maryland; Department of Neurobiology, and *The Jackson Laboratory, Bar Harbor, Maine Harvard Medical School, Boston, Massachusetts; Department of Dermatology, Southern General The asebia (ab) mouse mutation is a single gene recessive mutation that has been fine mapped to Hospital, Glasgow, UK; Department of Dermatology, Royal Victoria Infirmary, Newcastle upon mouse Chromosome 19 by genetic linkage analysis (Eilertsen et al., submitted). The mutant mice Tyne, UK; Department of Dermatology and Cutaneous Biology, Jefferson Medical College, display a phenotype consisting of hypoplastic sebaceous glands, alopecia, scaly skin, and lipid Philadelphia, Pennsylvania abnormalities. In order to identify the mutated gene a positional candidate gene approach was Recent advances provide the first evidence for the involvement of connexin defects in disorders initiated. Mutations were found in the SCD1 gene in two different strains of mice that are allelic of cornification. We identified mutations in GJB3 encoding the gap junction protein connexin- at the ab locus (abJ and ab2J). RT-PCR analysis in the DBA/1LacJ-ab2J/ab2J strain identified three 31 (Cx31) as the proximal cause of erythrokeratodermia variabilis, a dominant genodermatosis abnormal transcripts, with premature termination codons, that arise apparently from the use of that may present with palmoplantar keratoderma (PPK). Previously, a putative mutation of GJB2 cryptic splice sites. No SCD1 mRNA was detected in the skin of either mutant strain by northern (Cx26) was identified in one family with both PPK and deafness (PPK/DFN), although this analysis. SCD1 expression is limited only to sebaceous glands in the normal mouse skin as revealed variant was later suggested to be merely a nondisease associated polymorphism. In contrast, Cx26 by RNA in situ hybridization. The steady-state level of SCD1 mRNA in the sebaceous gland defects have been well established as the basis of nonsyndromic recessive (DFNB1), dominant varies over the induced hair cycle in adult mice. Expression of other members of the SCD family (DFNA3) and sporadic deafness. We studied two families with PPK/DFN consistent with dominant (SCD2 and a novel member, SCD3) in the mutant and normal mouse skin have also been studied. inheritance. In an Egyptian family the phenotype segregated with a heterozygous missense mutation Our results show that the defective SCD1 gene is responsible for the mouse asebia phenotype. of GJB2, leading to a nonconservative amino acid substitution (R75W). In a British family, we Although we do not yet fully understand the precise role of SCD1 in normal pilosebaceous identified in a father and son with PPK/DFN a heterozygous in-frame deletion that is predicted biology, the fact that the null SCD1 mutation (asebia) leads to profound cutaneous pathology, to eliminated E42. Moreover, the son was also heterozygous for 35delG, the most common suggests that this gene plays a very important, previously unappreciated, role in skin. recessive mutation of GJB2 in DFNB1 leading to premature protein termination. He inherited this deletion from his deaf mother who had normal skin, and was found to be homozygous for 35delG. These data suggest that not the recessive 35delG, but R75W and del42E are causally involved in PPK/DFN. Results of our coexpression studies of wild-type and mutant Cx26 in paired Xenopus oocytes further support this hypothesis. R75W alone was incapable of inducing electrical conductance between adjacent cells, and moreover, it almost completely suppressed the activity of coexpressed wild-type protein, demonstrating a deleterious dominant inhibitory effect of this Cx26 mutation on gap channel function consistent with the dominant inheritance. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 551

169 170 Mutant Loricrin is Not Crosslinked into the Cornified Cell Envelopes but Translocated into the Transgenic Mice Expressing a Mutant Form of Loricrin exhibit an Erythrokeratoderma similar to Nuclei in Loricrin Keratoderma Vohwinkel Syndrome A. Ishida-Yamamoto, H. Takahashi and H. Iizuka Y. Suga, H. Ogawa, D. Bundman and D. Roop Department of Dermatology, Asahikawa Medical College, Asahikawa, Japan Department of Dermatology, Juntendo University School of Medicine, Tokyo, Japan; Department We have recently found heterozygous mutations in the loricrin gene in two inherited skin diseases, of Cell Biology and Dermatology, Baylor College of Medicine, Houston, Texas Vohwinkel syndrome and progressive symmetric erythrokeratoderma (PSEK) and proposed to call Mutations in the cornified envelope protein loricrin have recently been reported in some families them loricrin keratoderma. These diseases are inherited as an autosomal dominant trait and the with Vohwinkel Syndrome (VS) and progressive symmetric erythro-keratoderma. We have patients are expected to express both wild and mutant loricrin molecules. Therefore the mutant generated transgenic mice expressing a mutant form of loricrin which is similar to that found in loricrin is likely to have a dominant negative effect. To understand the nature of this effect, we VS patients. We engineered a 1-bp insertion ofaCatnucleotide position 1190 in the mouse assessed localization of mutant loricrin molecules using antibodies raised against synthetic peptides loricrin coding sequence. This results in a frame shift and delayed termination, with replacement corresponding to the sequences unique to the mutant loricrin. Biopsy samples from two patients of the 86 carboxy-terminal amino acids by missense amino acids and an extension of 22 additional with PSEK were processed for light and electron microscopic immunohistochemistry. Patient residues. This VS mutant loricrin construct was introduced into the 6.5 kb mouse loricrin skin, but not normal control skin showed positive immunoreaction, which was abolished by expression vector (ML.VS) and injected into fertilized mouse embryos. At birth, the ML.VS absorbing the antibody with the mutant loricrin peptides. Mutant loricrin is localized in the nuclei transgenic mice exhibited a neonatal erythrokerato-derma, suggesting epidermal barrier dysfunction. of the differentiated epidermal cells. No substantial immunoreaction was observed on the cornified We measured increases in transepidermal water loss (TEWL) using Tewa MeterTM and the extent cell envelopes. The antibodies also stained parakeratotic nuclei of the scraped horny layer of the of TEWL was correlated with the severity of the phenotype. At about 7 days, highly expressing patient’s sole skin. These results strongly suggest that mutant loricrin does not directly disrupt transgenic animals showed a constricting band encircling the tail. Histologically, ML.VS transgenic crosslinking of cornified cell envelope precursors on the plasma membrane, but alters a late stage mice showed a marked parakeratosis with retention of nuclei in the stratum corneum. Immuno- of epidermal differentiation by interfering some nuclear functions. In addition, detection of the fluorescence analysis confirmed that the mutant loricrin protein was present in both the nucleus mutant loricrin in scraped horny layer could offer a simple noninvasive screening test for and cytoplasm of cells in the granular layer and the stratum corneum. A similar distribution of loricrin keratoderma. loricrin was previously observed in VS patients. This suggests that the VS mutant form of loricrin can exert a dominant, disruptive effect on cell envelope formation and may change its localization within the epidermis.

171 172 The Spectrum of GJB3 Mutations in Erythrokeratodermia Variabilis High Frequency of Allelic Deletion on 9p21 (p16 Gene Locus) in Melanoma Metastases of Non- G. Richard, G. Melino,* J. DiGiovanna† and S. Bale‡ Responders to Peptide-Pulsed Dendritic Cell Vaccination Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Philadelphia, R. Bo¨ni, D. Matt, H. Xin, F. O. Nestle, M. Gilliet and G. Burg Pennsylvania; *Laboratory of Biochemistry, IDI-IRCCS, Rome, Italy; †Department of Dermato- Department of Dermatology, University Hospital, Zu¨rich, Switzerland logy, Brown University/Rhode Island Hospital, Providence, Rhode Island; ‡Genetic Studies Peptide- or tumor lysate-pulsed dendritic cell vaccination is a promising approach to the treatment Section, NIAMS, NIH, Bethesda, Maryland of metastatic melanoma (Nature Medicine 1998; 4:328–332). Intercellular channels in skin are a complex and functionally diverse system formed by at least To elucidate the role of genetic alterations of metastatic melanoma in nonresponders to vaccination seven connexins (Cx) including Cx31. Although their role in epidermal differentiation is still therapy, we performed a microdissection based genetic analysis of the tumor suppressor gene p16. poorly understood, results of our molecular studies implicate for the first time connexin defects One metastasis in a patient showing complete response (one of 10), two metastases in two patients in an inherited disorders of cornification. Erythrokeratodermia variabilis (EKV) is an autosomal with stable disease (two of 10), and seven metastases in seven patients with progressive disease dominant genodermatosis with a striking phenotype characterized by the independent occurrence (seven of 10) were exzised and analysed. Two to five different areas within individual metastases of two morphologic features: localized erythema and hyperkeratosis, which can be triggered by as well as adjacent normal tissue were microdissected and subjected to single step DNA extraction. external factors such as trauma to the skin. We mapped EKV to 1p34-p35, and identified the Extracted genomic DNA was amplified by polymerase chain reaction using two polymorphic positional candidate gene GJB3 encoding Cx31. Mutation analysis of GJB3 revealed four different markers on 9p21 (p16 gene locus: IFNA, D9S171). heterozygous missense mutations in five of 13 unrelated EKV families. Two mutations affected a There was a concordance of genetic changes within different areas of individual metastases. Allelic phylogenetically conserved glycine in the N-terminus of Cx31 (G12R, G12D), while the other deletion was retained in the three patients with complete response/stable disease, while in four of mutations lead to nonconservative substitutions in the first extracellular loop (R42P) and in the seven patients with progressive disease, loss of heterozygosity could be demonstrated for IFNA second transmembrane domain (C86S). In one family, all affected individuals were compound and D9S171. hetero-zygotes for R42P and an in frame-deletion eliminating four residues of the C-terminus. These data show a homogeneity of genetic events within melanoma metastases and indicate a However, the deletion was present in several unaffected family members suggesting it is not strong role of the p16 tumor suppressor gene in the progression of metastatic melanoma. Analysis associated with EKV. The presence of an identical mutation in two families with diverse phenotypic of the p16 gene might be useful to value the effect of current vaccination trials. features indicates that other factors might be responsible for the clinical variability of the disease. In contrast to a report linking Cx31 mutations to progressive hearing impairment (Xia et al., Nature Genetics 20:370–373, 1998), none of our families with Cx31 mutations were affected with this condition. All our missense mutations are predicted to interfere with normal Cx structure/ function, possibly due to dominant inhibition of normal Cx channel activity by affecting assembly, transport, or channel properties. The altered Cx31-mediated communication between coupled cells may lead to disturbed differentiation (keratinocytes) and vascular dilatation (capillaries) characteristic for EKV. Our studies implicate Cx31 in the pathogenesis of EKV, and provide the first evidence that intercellular communication mediated by Cx31 is crucial for epidermal differentiation and response to external factors.

173 174 Systemic Delivery of Human Transferrin from Genetically Modified 3T3 Cells Housed in an In Vitro and In Vivo, Expression of the Full-Length cDNA of Type VII Collagen; Possibility of Encapsulated Membrane in the Subcutaneous Space of Athymic Mice Gene Therapy for Dystrophic Epidermolysis Bullosa (DEB) P. A. Tresco, B. W. Hitchcock, C. M. Jorgensen and G. G. Krueger D. Sawamura, X. Meng, K. Itai, S. I. A. Kon, K. Tamai, H. Hanada, T. Tanaka* and I. Hashimoto W M Keck Center for Tissue Engineering and the Departments of Bioengineering and Departments of Dermatology, Hirosaki University School of Medicine and *Graduate School of Dermatology, University of Utah, Salt Lake City, Utah Medicine, Kyoto University, Kyoto, Japan The use of encapsulated devices to house cells to deliver bioactive molecules has focused on DEB is an inherited disorder caused by mutations in COL7A1. Introduction of this gene to approaches to provide controlled, sustained delivery of therapeutic substances for the treatment of keratinocytes is now expected for potential therapy of DEB. In this study, we first constructed selected disorders. Genetically engineered autologous cells and or stem cells may one day preclude the 9 kb full-length cDNA from cDNA clones of COL7A1. After sequence analysis, we subcloned the need for the immunoshielding function of encapsulated systems. Nevertheless, an easily the full-length cDNA into pCY4B expression vector and transfected it to mouse keratinocytes retrieved device that physically isolates the transplanted cells from the host tissue has value as a (Pam cells). Expression of transgenic protein was confirmed by antibodies against the NC1 and 2 research tool for evaluating the clinical potential of a variety of cell replacement therapies. domains of the protein. Furthermore, we subcloned the cDNA to a vector of green fluorescent Encapsulation membranes were developed that were nontoxic to 3T3 cells that had been transduced protein (GFP) to produce type VII collagen-GFP fusion protein. Transfection of this construct to with a retroviral vector encoding human transferrin (hTf) and cloned for hTf production. These Pam cells found that localization of the fusion protein in the cells was similar to that of type VII cells were loaded into these encapsulation membranes and implanted to the subcutaneous space collagen of normal keratinocytes. Next, we deleted the cDNA from the 3’- and constructed of six athymic mice for 14 d. Serum levels of hTf were measured by ELISA. The mean hTf levels several mutant-GFP proteins, resulting in change of localization of the fusion proteins. Finally, were 137 ng per ml at day 7 and 223 ng per ml day 14, no detectable levels of hTf were noted we introduced the full-length cDNA into rat keratinocytes in vivo using the naked DNA method in the controls, 3T3 cells transduced with a lacZ retroviral vector. The implants were retrieved at and found expression of the protein. This full-length cDNA will provide further investigation of day 14 and the cells isolated for regrowth in tissue culture. There were no signs of toxicity and this protein and much contribution to fundamental study of DEB gene therapy the recovered cells produced hTf levels of the nontransplanted counterparts. These findings indicate that this system has significant potential as a research tool and, possibly as a therapeutic approach to deliver large biologically active molecules. 552 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

175 176 Corrective Gene Transfer in Dystrophic Epidermolysis Bullosa A Raft Culture Model Demonstrates the Feasibility of Topical Selection for Keratinocytes M. Chen, E. A. O’Toole, Y-Y. Liu, N. Kasahara and D. T. Woodley Expressing a Desired Gene Department of Dermatology, North-western University of Chicago, Illinois; Departments of W. Pfu¨tzner, M. A. Joari, U. R. Hengge* and J. C. Vogel Pathology and Biochemistry, University of Southern California, Los Angeles, California Dermatology Branch, NCI, National Institutes of Health, Bethesda, Maryland; *Department of Dystrophic epidermolysis bullosa (DEB) is a family of inherited mechano-bullous disorders caused Dermatology, University of Essen, Germany by mutations in the type VII collagen gene (COL7A1) and subsequent perturbations in anchoring In gene therapy studies, persistent expression of an inserted gene has been problematic. One fibrils. The lack of therapy for DEB provides an impetus to develop gene therapy strategies. We solution to enhance expression in skin gene therapy would be to topically select for keratinocytes previously constructed a recombinant type VII collagen ‘‘minigene’’, comprising the sequences (KC) expressing both the desired gene and a linked selectable marker gene. The multi drug encoding for the entire noncollagenous globular domains (NC1 and NC2, respectively) and half resistance (MDR) gene confers resistance to a variety of cytostatic and antimitotic compounds, of the central helical collagenous domain, minus a 2-kb in frame deletion. Functional analyses such as colchicine. Utilizing a KC raft culture model to test feasibility of this approach, primary showed that the ‘‘minigene’’ product retained the function and characteristics of a full-length type human KC were transduced with the MDR retroviral vector pHaMDR1/A (transduction VII collagen alpha chain. In this study, we used a high-efficiency retroviral delivery approach to efficiency up to 75% by flow cytometry analysis), grown on acellular dermis, and treated with transduce RDEB keratinocytes (in which the expression of type VII collagen was absent) with colchicine. The ability of MDR-expressing KC to proliferate and to establish a stratified, the ‘‘minigene’’ construct. Minigene-transduced RDEB keratinocytes synthesized and secreted a differentiated epidermis under colchicine selection were investigated. At different colchicine doses 240-kDa recombinant type VII ‘‘minicollagen’’ at levels equivalent or higher than normal cells. (10–50 ng per ml), MDR-transduced KC were able to form a well-differentiated epidermis as The expression of the ‘‘minicollagen’’ was sustained for at least 6 mo. Gene-corrected RDEB assessed immunohistochemically by KC differentiation markers while KC transduced with a keratinocytes demonstrated enhanced cell-substratum adhesion and increased proliferative potential. retroviral control vector did not proliferate and developed an epidermis. During colchicine The gene-corrected cells also showed reversal of the hypermotility phenotype characteristic of selection, the MDR-expressing KC had the same proliferative capacity in the rafts cultures as uncorrected RDEB cells. Further, we also isolated RDEB dermal fibroblasts which completely untreated control KC, as shown by immunohistochemistry and flow cytometry analysis for lacked type VII collagen expression. Transduction of the RDEB fibroblasts with the retroviral proliferating cell nuclear antigen (PCNA) expression. Significantly, colchicine treatment increased ‘‘minigene’’ construct resulted in synthesis and secretion of the ‘‘minicollagen’’ which was the percentage of KC expressing MDR (almost 100%) in raft cultures, by both immunohistochem- maintained at stable levels for at least 4 mo. Gene correction fully restored the RDEB fibroblast’s istry and flow cytometry analysis. This raft culture model demonstrates that MDR-expressing KC proliferative potential. These data indicate that efficient ‘‘minigene’’ delivery to keratinocytes and can proliferate and be enriched by colchicine selection, yet are still able to stratify properly and fibroblasts can produce a population of phenotypically corrected RDEB skin cells. Because cultured form a differentiated epidermis. Similar in vivo applications in an animal model should be feasible keratinocytes are used routinely to make autologous grafts for burn patients, the capability to and are currently being conducted. Topical selection should prolong and enhance expression of a produce gene-corrected skin cells may provide a basis for future ex vivo gene therapy for DEB. desired gene linked to the resistance gene and offer new possibilities for long-term treatment in skin gene therapy.

177 178 Transfection of Human Hair Follicles Using Topical Liposomes is Optimal at the Onset of Anagen Stable and Inheritable Changes in Pigmentation and Chromosome of Albino Melanocytes by an A. Domashenko and G. Cotsarelis RNA-DNA Oligonucleotide Department of Dermatology, University of Pennsylvania, Philadelphia, Pennsylvania V. Alexeev and K. Yoon Although topical liposome preparations can deliver plasmid DNA to mouse hair follicles, human Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Philadelphia, hair follicles have not been transfected with this approach. We demonstrate that liposome Pennsylvania application at the onset of anagen is critical for successful transfection of both mouse and human Recent studies have shown the development of experimental strategies to correct point mutations hair follicles. We screened eight different liposome preparations for their ability to transfect a beta by chimeric oligonucleotides composed of RNA and DNA (RDO). We applied this method to galactosidase reporter plasmid into mouse skin in vivo and human hair follicles in organ culture. correct a point mutation in the mouse tyrosinase, a key enzyme for melanin synthesis and The same liposome preparation yielded the highest transfection efficiency in both systems. We pigmentation. Melanocytes derived from albino mice contain a homozygous point mutation then used this liposome preparation to topically deliver a reporter plasmid to human scalp grafted (TGT001→TCT) in the tyrosinase gene, resulting in an amino acid change from Cys001→Ser. to immunodeficient mice. The beta galactosidase reporter was expressed only in hair follicles just Correction of this point mutation results in the restoration of tyrosinase activity and melanin entering anagen (stage I–IV). No follicles in full anagen (stage VI) or telogen were transfected. synthesis, thus changing the pigmentation of the cells. Upon transfection of the RDO to albino Transfected cells primarily consisted of newly forming matrix keratinocytes located at the leading melanocytes, we detected black-pigmented cells and isolated multiple single clones. All black- edge of the downgrowth. Hair plucking increased the percentage of follicles in the early anagen pigmented clones exhibited a correction of the point mutation in a single allele of the tyrosinase stage (from 1% to 14%), and 11% (five of 45) of these early anagen follicles were transfected for gene. A full-length tyrosinase was detected by an antityrosinase antibody and the enzymatic activity an overall transfection rate of 1.6% (five of 318). Combined plucking and retinoic acid application was restored in all converted black-pigmented clones. The phenotype and genotype of converted increased the frequency of follicular transfection to 5.9% (26 of 438) of all follicles, and 49% (26 black-pigmented clones was stable. These results demonstrate for the first time a permanent and of 53) of follicles entering anagen. We conclude that hair follicles are particularly susceptible to stable gene correction by the RDO at the level of genomic sequence, protein, and phenotypic transfection with plasmid DNA using topical liposomes during anagen onset. Because most types change by clonal analysis. The delivery of RDO into BALB/C mice was carried out by a topical of alopecia are characterized by an increased percentage of telogen follicles that can be stimulated application of RDO with liposomes or intradermal injection. Skin biopsies taken from the treated into anagen (as in androgenetic alopecia) or of an increased percentage of early anagen follicles mice indicate a pigmentation change of a few hair follicles, albeit at a low frequency. (as in alopecia areata), our results suggest that these alopecias may be amenable to gene therapy.

179 180 SMAD-Dependent Regulation of Type I Collagen Gene Expression Retinoic Acid Receptor (RAR)-but not Retinoid X Receptor (RXR)-Selective Retinoids H. Nakano, L. Vindevoghel, D. J. Kouba, J. Uitto and A. Mauviel Suppress Ultraviolet Light (UV) Induction of c-Jun Protein Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Jefferson Institute J. Xiao, T. He, H. Talwar, W. Di, E. Duell and J. Voorhees of Molecular Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania Department of Dermatology, University of Michigan, Ann Arbor, Michigan We have previously identified the TGF-001β-response element (TBRE) of the human alpha2(I) In cultured cells, retinoids such as all-trans retinoic acid (tRA) enable their receptor to interact collagen gene (COL1A2) promoter within the region between nucleotides –271 and –235. Thus with transcription coactivators, resulting in target gene activation, and sequester shared coactivators far, however, the exact nature of the transcription factors binding the TBRE has not been from transcription factor complex AP1, causing repression of AP1-target genes. In vivo,UV elucidated. Using electrophoretic mobility shift assays (EMSAs), we have now identified a single irradiation of human skin activates c-Jun, an AP1 component, which induces transcription of dermal protein/DNA complex rapidly induced by TGF-001β, with a kinetic of appearance consistent matrix-degrading metalloproteinase, ultimately leading to photoaging. Topical tRA suppresses UV with that of the recently described SMAD proteins, key elements of the TGF-001β immediate induction of c-Jun protein. As RAR-selective tRA is isomerized to 9-cis RA, an RAR/RXR signaling mechanisms. Furthermore, comparison of the nucleotide sequence of the COL1A2 pan-agonist in skin cells, it raised the question as to whether the tRA suppression of c-Jun is TBRE with that of published SMAD binding sequences revealed some striking similarities, and mediated by RAR or RXR. To investigate this possibility, skin of C57BL/6 mice was treated specifically, the COL1A2 promoter TBRE contains a CAGA box flanking an AP-1-like element. with tRA or receptor-selective synthetic retinoids and then irradiated with UV (290–400 nm, These results led us to investigate the role of SMADs on COL1A2 promoter activity. Using 001λmax ϭ 310 nm). Epidermal c-Jun was measured by western analyses. UV dose-dependently expression vectors for the SMAD members involved in TGF-001β signaling, namely SMAD2, (25–150 mJ per cm2) induced c-Jun protein. Maximal induction of c-Jun protein (8.4 001Ϯ 1.1- SMAD3 and SMAD4, we demonstrate that SMAD2 and SMAD3 activate COL1A2 promoter fold, n ϭ 12, p Ͻ 10–4) was observed at 6 h, post-UV irradiation (150 mJ per cm2, equivalent to activity in an additive manner whereas SMAD4 alone has no effect. Electrophoretic mobility shift 3 MED in humans). 48-h tRA (40 nmol) pretreatment suppressed UV induction of c-Jun protein assays indicate that both recombinant SMAD3 and SMAD4 have the ability to bind the COL1A2 by 89% (n ϭ 12, p Ͻ 10–4), but not c-Jun kinase activity. An RAR-selective and 4-hydroxylase- TBRE. Together, these data indicate that the SMAD signaling pathway is involved in the resistant retinoid, CD367 (0.4 nmol), suppressed UV induction of c-Jun protein by 83% (n ϭ 8, regulation of type I collagen gene expression and indicate that targeting SMADs may be of interest p Ͻ 10–4). RXR-selective retinoid SR11237 (20 nmol) had no effect (n ϭ 5). Prolonged towards pharmacologic manipulation of fibrotic disorders in which collagen accumulation is likely pretreatment (Ͼ24 h) was necessary for tRA and CD367 to be effective, indicating that the due to TGF-001β overexpression. suppression requires multistep molecular changes in epidermal cells. Taken together, our data suggest that in skin in vivo, the ability of tRA to prevent UV induction of c-Jun is mediated by RARs. Thus, RAR-but not RXR-selective synthetic retinoids may be used to prevent skin photoaging. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 553

181 182 Regulation of Involucrin Promoter Activity by Specific Protein Kinase C Isoforms – PKC- Involucrin Promoter-dependent Expression of Human Amphiregulin cDNA in Murine Suprabasal Dependent Upregulation of AP1 Epidermis Produces a Psoriasis-like Cutaneous Pathology T. Efimova and R. Eckert P. W. Cook, J. R. Brown and M. R. Pittelkow† Physiology & Biophysics, Dermatology, Biochemistry, and Oncology, Case Western Reserve Department of Dermatology, Oregon Health Sciences University, Portland, Oregon; and *Depart- University School of Medicine, Cleveland, Ohio ment of Dermatology Mayo Clinic, Rochester, Minnesota Involucrin (hINV) mRNA level and promoter activity increase when keratinocytes are treated Amphiregulin (AR) is a heparin-regulated EGF family member whose expression is upregulated with the differentiating agent, phorbol ester (TPA). This activation is mediated via a p38 and in the epidermis of tape-stripped skin, skin organ cultures, cultured keratinocytes, developing activator protein 1 pathway. In the, present study we examine the role of various PKC isoforms epidermis and in the epidermis of proliferative skin pathologies (psoriasis, AK, SCC, verrucae) in this regulation. Transfection of expression plasmids encoding PKC001δ, 001ε, and 001η We have previously shown that keratin 14 promoter-dependent transgenic expression of the the increases hiNV promoter activity and expression of the endogenous hiNV gene as efficiently as human AR gene (K14-ARGE) results in lethality, and produces a cutaneous phenotype closely TPA. In contrast, PIKC001α, 001β, 001γ, and 001ζ do not increase promoter activity. This resembling that of lesional psoriatic skin. In an attempt to determine whether suprabasal expression PKC001δ, 001ε- and 001η-dependent activation is inhibited by dominant-negative forms of Ras, of AR might produce a similar cutaneous phenotype in the absence of lethality, we expressed an MEKK1, p38 or AP1, but not by dominant-negative Raf-1 or JNK1. The PKC001δ-selective Involucrin promoter-driven AR transgene (INV-AR) in mice. Analysis of founder INV-AR mice inhibitor, rottlerin, also inhibits activation. This regulation appears to target an AP1 site in the demonstrated expression of human AR in suprabasal epidermis and revealed a phenotype which proximal regulatory region (PRR) of the hINV promoter. JunB, JunD and Fra-1 form a complex was similar to that of K14-ARGE mice. This included poor viability, generalized scaling and that binds at this AP1 site. Activation is.associated with increased immunodetectable JunB, JunD erythematous skin with alopecia. Histologic examination revealed focal parakeratosis, hyperkeratosis, and Fra-1 and increased AP1 factor binding at the hINV gene AP1Ϫ1 site. Moreover, the acanthosis, T-cell infiltration, and an exaggerated dermal vasculature. Our results corroborate our composition of AP1 factors present at the hINV AP1Ϫ1 site changes in TPA-treated cells. These earlier findings and suggest that AR exerts activities in the skin which are different from that of results suggest that involucrin gene expression is mediated via a pathway that involves novel PKC TGF001α, other growth factors or inflammatory cytokines, and that aberrant expression of specific isoforms but not conventional PKC001α, 001β, 001γ or atypical PKC001ζ, and that activation EGF-related ligands and receptors in the epidermis may represent an important step in the results in increased AP1 factor levels and AP1 factor binding at a specific AP1 site in the hINV development of psoriatic lesions. gene proximal regulatory region.

183 184 A New Role for p75 Neurotrophin Receptor in Hair Follicle Regression: Catagen Retardation Inhibition of MAP Kinase Kinase Causes Morphologic Reversion and Dissociation Between Soft in p75NTR Knockout Mice and After p75NTR Blockade by Cyclic Peprides Agar Growth and In Vivo Tumorigenesis in Angiosarcoma Cells V. A. Botchkarev,*† N. V. Botchkareva,* M. Yaar,† B. A. Gilchrest† and R. Paus* J. L. Arbiser,K. R. LaMontagne Jr, M. A. Moses, D. Wiederschain, S. Mahajan, H. Ghazizadeh *Department of Dermatology, Humboldt University, Berlin, Germany; and †Department of and D. A. Frank Dermatology, Boston University School of Medicine, Massachusetts Department of Dermatology, Emory University School of Medicine, Atlanta, Georgia; Department We have recently shown that neurotrophins are involved in the control of apoptosos-driven hair of Surgical Research, Childrens Hospital, Boston, Massachusetts follicle (HF) regression (catagen). In order to examine the role of p75NTR, implicated in apoptosis Activated ras causes increased activity of several signal transduction systems, including the MAP control, the expression and function of p75NTR during spontaneous catagen development was kinase kinase (MAPKK) pathway and phosphoinositol-3-kinase (PI-3-K) pathways. We have studied in murine skin. Using immunohistochemistry, we found that p75NTR alone is strongly previously shown that the PI-3-K pathway plays a major role in regulation of ras mediated tumor expressed in keratinocytes (KC) of the regressing outer root sheath, but both p75NTR and TrkB angiogenesis in angiosarcoma. However, the contribution of the MAPKK pathway to tumorigenesis and/or TrkC are expressed by the nonregressing secondary hair germ KC. Because p75NTR and angiogenesis is not fully understood. To study the effect of MAPKK downregulation on signals for apoptosis when activated alone, but instead for survival when activated together with tumorigenesis and angiogenesis, we introduced a dominant-negative MAPKK gene into SVR receptors of the Trk family, we examined the correlation between HF KC apoptosis and p75NTR/ murine angiosarcoma cells, which are derived from murine endothelial cells through the sequential Trk expression. TUNEL positive HF KC expressed only the p75NTR, while the surviving introduction of SV40 large T antigen and activated H-ras. Introduction of a dominant negative secondary hair germ KC expressed in addition Trk receptors. To determine if p75NTR is MAPKK causes a significant decrease in proliferation rate in vitro and morphologic reversion. Cells functionally involved in catagen control, spontaneous catagen development was compared in vivo containing the dominant negative MAPKK grew 75% slower in tissue culture, and their growth between p75NTR knockout (– /–) and age-matched wild type mice. There was a significant was not inhibited by the MAPKK inhibitor PD98059. Surprisingly, cells overexpressing dominant catagen retardation by 12% in p75NTR knockout mice as compared to wild type controls (p Ͻ negative MAPKK or treated with the MAPKK inhibitor PD98059 showed increased expression 0.05). To further examine the role of p75NTR in catagen development of C57BL/6 mice, we of matrix metalloproteinases. Cells expressing the dominant negative MAPKK have a greatly supplemented HF catagen organ cultures with diluent alone or with cyclic peptides known to decreased ability to form colonies in soft agar compared with wild type cells. Despite the decreased bind the p75NTR and previously shown to block apoptosis in other cell types that require cell growth in vitro and inability to grow in soft agar, the cells were equally tumorigenic in nude p75NTR activation. Cyclic peptides (0.01–100 µM) significantly retarded catagen development mice. To our knowledge, this is the first example of a cell line which is tumorigenic but has by 33% compared to vehicle control (p Ͻ 0.05). Our findings suggest that p75NTR signalling is impaired ability to grow in soft agar. These findings have implications in the development of involved in the control of KC apoptosis during catagen and that p75NTR antagonistic cyclic signal transduction modulators as potential antineoplastic agents. peptides may prove useful for the treatment of hair disorders that display premature entry into catagen (e.g. telogen effluvium, androgenetic alopecia, alopecia areata).

185 186 Distinct Roles of Extracellular Signal-Regulated Kinase and p38 Mitogen Activated Protein Epithelial Growth Regulation by Reciprocal Acting Cytokines in Organotypic Fibroblast- Kinase in Activation of Collagenase-1 (MMP-1) and Stromelysin-1 (MMP-3) Expression in Keratinocyte Cocultures Dermal Fibroblasts N. Maas-Szabowski and N. Fusenig N. Reunanen, M. Foschi, J. Han and V.-M. Ka¨ha¨ri German Cancer Research Center, Heidelberg, Germany Department of Dermatology, University of Turku, Finland; University of Firenze, Italy; and Dermal–epidermal interactions regulate keratinocyte proliferation, differentiation and tissue recon- Scripps Research Institute, La Jolla, California struction via complex paracrine mechanisms. These interactions were studied in an in vivo like We have examined the role of mitogen-activated protein kinase (MAPK) signaling pathways in model with normal human epidermal keratinocytes growing on irradiated postmitotic dermal the regulation of collagenase-1 (matrix metalloproteinase-1, MMP-1) and stromelysin-1 (MMP- fibroblasts embedded in a collagen type I matrix. These postmitotic cells, mimicing the state of 3) expression in normal human skin fibroblasts. Tumor necrosis factor-001α (TNF-001α) and resting fibroblasts predominant in dermis, maintain basal and inducedmRNA-expression and interleukin-1001β (IL-1001β) are inflammatory cytokines, which induce MMP-1 and MMP-3 protein synthesis of various cytokines. In both cell types, the expression levels of several cytokines expression in these cells. TNF-001α and IL-1001β activate the extracellular signal-regulated kinase in mono-and coculture were determined by semiquantitative RT-PCR and western blot or ELISA. (ERK)1,2, as well as the stress-activated protein kinase/Jun-N-terminal kinase (SAPK/JNK) and In organotypic cocultures, mRNA levels of KGF, TGF-001α, GM-CSF and IL-1RI are much p38 MAPK in these cells. TNF-001α- and IL-1001β-elicited induction of MMP-1 and MMP-3 higher in cocultured than in monocultured fibroblasts in collagen gels, whereas HGF and TGF- mRNA was inhibited by a specific p38 MAPK inhibitor SB203580. However, blocking the 001β1-expression is not affected. In keratinocytes growing on fibroblast containing gels, the ERK1,2 pathway (Raf/MEK1,2/ERK1,2) by MEK1,2 activation inhibitor PD98059 had no expression of IL-1 and KGF-receptor is increased as compared to monocultures. effect on the TNF-001α-or IL-1001β-induced expression of MMP-1 and MMP-3. Infection of IL-1 does not stimulate keratinocyte proliferation in monoculture, but induces in fibroblasts the cells with recombinant adenovirus harboring constitutively active MEK1 resulted in activation increased expression of KGF, HGF, TGF-001α, and GM-CSF, growth factors which stimulate of ERK1,2, and markedly enhanced the mRNA levels and production of MMP-1 and MMP-3. keratinocyte proliferation. Thus, we postulated that IL-1 indirectly stimulates keratinocyte growth Infecting cells with an adenovirus for a constitutively active MKK6b (p38 kinase) specifically by a double paracrine pathway. activated p38 MAPK and induced the expression of MMP-3, but had no effect on MMP-1 Consequently, addition of IL-1 neutralizing antibodies or IL-1-receptor antagonist to organotypic expression. Adenovirus-mediated expression of dominant negative MKK6b potently inhibited cocultures inhibits epithelial growth. Furthermore, the reduced fibroblast support of subthreshold activation of p38 MAPK and upregulation of MMP-1 and MMP-3 production by TNF-001α fibroblast numbers could be compensated by adding IL-1 to the culture medium. Thus, IL-1, and IL-1001β. These results show that although the activation of ERK1,2 pathway alone is being one or the main signal transducer between epidermal and dermal cells indirectly stimulates sufficient for the induction of MMP-1 and MMP-3 expression, the stimulatory effects of TNF- keratinocyte proliferation by inducing growth factor expression in cocultured fibroblasts. 001α and IL-1001β on MMP-1 and MMP-3 expression in dermal fibroblasts require activation of p38 MAPK. Thefore, inhibition of p38 pathway may have therapeutic importance in inflammatory conditions characterized by excessive degradation of collagenous extracellular matrix. 554 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

187 188 Early Gene Responses in Human Skin Organ Culture are MAP Kinase-Dependent, Whereas Oncostatin M Stimulates the Growth of Dermal Fibroblasts via a Mitogen-Activated Protein Keratinocyte Outgrowth is p38-Dependent Kinase-Dependent Pathway S. Stoll and J. Elder H. Ihn and K. Tamaki Department of Dermatology, University of Michigan, Ann Arbor, Michigan Department of Dermatology, Faculty of Medicine, University of Tokyo, Tokyo, Japan Reepithelialization (RE) is a highly coordinated response to injury involving dynamic alterations Oncostatin M (OSM), a member of the hematopoietic cytokine family, has been implicated in in keratinocyte (KC) migration, proliferation, and differentiation. Many aspects of RE are the process of fibrosis. As part of an ongoing study of the mechanisms of fibrosis, we have recapitulated in skin organ culture (JCI 100:1271). Therefore, organ culture provides an ideal investigated the regulation of the growth of dermal fibroblasts by OSM. OSM stimulates the vehicle for the dissection of signal transduction pathways involved in RE. For assay of short-term mitogenesis of dermal fibroblasts in a dose-dependent manner. This effect was completely blocked (0.5–24 h) gene responses, human skin keratome fragments (001µ1cm2) were incubated in basal by anti-OSM antibody, but not by anti-IL-6 antibody. Furthermore, the mitogenic activity modified MCDB 153 at 37°C, followed by RNA isolation and northern blotting. For measurement induced by OSM was inhibited by genistein, a tyrosine kinase inhibitor, or by PD98059, a specific of epidermal outgrowth, explant outgrowth assays were perfomed as described previously (JCI mitogen-activated protein (MAP) kinase inhibitor, but not by calphostin C, a protein kinase C 100:1271). c-Fos and c-jun mRNA responses were observed after as little as 0.5 h of organ inhibitor. Further analysis showed that OSM induces tyrosine phosphorylation of MAP kinase in culture, whereas heparin-binding EGF-like growth factor (HB-EGF) and vascular permeability dermal fibroblasts. Moreover, transient transfection of dominant negative MAP kinase into factor (VPF) were not detectably induced until 1–2 h. All these responses were markedly inhibited fibroblasts inhibited the mitogenic activity induced by OSM. These results strongly suggest that (Ͼ80%) by 50 µM PD98059, a specific inhibitor of the mitogen-activated protein kinase (MAPK) OSM stimulates the growth of dermal fibroblasts via a MAP kinase-dependent pathway. pathway (JBC 270:27489). By 24 h of organ culture, c-fos and c-jun mRNAs returned to near- background levels, whereas HB-EGF and VPF remained strongly induced. However, 50 µM PD98059 no longer inhibited any of these later mRNA responses, despite being present for the entire 24-h period. This result suggested that other signal transduction pathways become important as RE progresses. Consistent with this interpretation, PD98059 had no effect on KC migration or proliferation in explant outgrowth cultures. However, KC migration (1–3 d) and proliferation (Ͼ3 d) were profoundly inhibited by SB202190, a specific inhibitor of p38. This dual function kinase is related to yet clearly distinct from MAPK. These results suggest that mechanisms for switching between alternative signal transduction pathways exist in KC and play a major role in the control of RE.

189 190 The TNF-001α Receptor-Associated Factors TRADD and TRAF2 Inhibit Type I Collagen gene The Upward Concentration Gradient of Free Acetylcholine in Human Epidermis Provides for its (COL1A2) Transcription in Human Dermal Fibroblasts Unopposed Secretagouge Action in the Stratum Granulosum: a Step Toward Terminal Differen- D. J. Kouba, A. Kon, J. Kang, L. Vindevoghel, J. Uitto and A. Mauviel tiation Departments of Dermatology and Cutaneous Biology, and Biochemistry and Molecular Pharma- A. Ndoye, V. Nguyen, B. Zelickson,* M. Lawry and S. Grando cology, Jefferson Medical College, Jefferson Institute of Molecular Medicine, Thomas Jefferson Departments of Dermatology, University of California, Davis, California; and *University of University, Philadelphia, Pennsylvania Minnesota, Minneapolis, Minnesota The TNF-001α Receptor (TNFR)-associated molecules TNF Receptor Associated Death Domain Terminally differentiated keratinocytes (KC) extrude their cytoplasm via zeiosis. This phase of KC protein (TRADD) and TNFR-Associated Factors (TRAF1, TRAF2 and TRAF3) mediate signals differentiation is similar to secretion and involves cell membrane blebbing. The secretagouge from the members of the TNF receptor superfamily. We and others have previously shown that involved remains unknown. KC synthesize acetylcholine (ACh), which acts as a secretagouge in TNF-001α suppresses transcription of the human COL1A2 gene in vitro, and we have recently the epithelium. In epidermis, ACh exhibits both the muscarinic and nicotinic effects which are identified NF-001κB as an essential transcription factor in this process (Kouba et al., J Immunol balanced by simultaneous activation of both receptor types and are opposed by acetylcholinesterase 1999, in press). Here, we have explored the role played by TRAF1–3 and TRADD in this (AChE), which, in addition to ACh receptors, is expressed on the cell membrane of KC. Since inhibitory effect. Our data indicate that the TNFR1 (p55) specific factor, TRADD, as well as the concentration of free ACh is determined by the ratios of its synthesis by choline acetyltransferase TRAF2, potently inhibit COL1A2 promoter activity, whereas TRAF1 and TRAF3 do not. Both (ChAT) and hydrolysis by AChE, we quantified the amounts of both enzymes and free ACh in TRADD and TRAF2 inhibit COL1A2 transcriptional activity independently of NF-001κB epidermis using an immuno-fluorescence assay. ChAT distributed equally throughout epidermis, activation. Promoter deletion analysis demonstrates that the inhibitory effect of TRAF2 localizes while AChE decreased from 111.1 Ϯ 4 to 40.5 Ϯ 5(pϽ0.05) during differentiation of KC from to a region of the minimal promoter within 108 bases of the transcription start site, whereas the basal to granular KC. The amount of free ACh increased accordingly from 8.7 Ϯ 2to59Ϯ3(p NF-001κB-mediated TNF-001α inhibitory effect localizes to – 265/–241. In addition, we have Ͻ 0.05). This upward gradient of free ACh can lead to its unopposed action in the superficial found TRADD is a cell-type specific modulator of NF-001κB activation. Specifically, TRADD layers of epidermis. To reproduce in vitro unopposed effects of ACh, KC monolayers were up-regulates NF-001κB-driven transcription in HeLa and 293 cells, as measured using reporter pretreated for 10 min with nicotinic or muscarinic antagonist and then exposed an AChE-resistant constructs, but down-regulates NF-001κB-dependent transcription in NIH 3T3 and human agonist. The nicotinic, but not muscarinic, stimulation made KC extrude into culture medium dermal fibroblasts. These data indicate that the TNFR signaling system acts on collagen gene globular blebs which grew in size (up to 10 µm) and number (up to 15 per cell) within 10 min. expression by mechanisms that are either utilizing NF-001κB or other, still unexplained, pathways These spheres were similar to those evolving from the dome-shaped blebs that project sporadically acting on the proximal region of the COL1A2 promoter. from terminally differentiated KC. Scanning electron microscopy revealed that at first the spheres are simple, but later their surface becomes dimpled and infolded. Thus, gradual disappearance of AChE from maturing KC, leading to unopposed action of ACh, can provide for zeiosis of terminally differentiated KC, and this secretagouge effect of ACh is mediated by activation of the nAChR expressed by granular cells.

191 192 Antimicrobial Activity of Granulysin: Mechanism of Action Expression of CLA-Epitope is Limited to PSGL-1 on Neutrophils, Monocytes and Blood Dendritic W. Ernst, D.A. Hanson, C. Ko, A. Krensky, C. Clayberger and R. Modlin Cells: A Common Theme for Construction of Leukocyte Selectin Ligands Division of Dermatology, UCLA, Los Angeles California; and Division of Immunology and J. Kieffer, R. Fuhlbrigge, D. Armerding, C. Robert and T. Kupper Transplantation Biology, Stanford University, Stanford, California Harvard Skin Disease Research Center, Department of Dermatology, Brigham and Women’s Granulysin, a protein located in the cytotoxic granules of human cytolytic T cells, has a broad Hospital, Boston, Massachusetts spectrum of activity against cutaneous pathogens, including bacteria, fungi and parasites. When The extravasation of memory T cells into skin has been associated with cell-surface expression of combined with the pore-forming protein, perforin, granulysin can also kill intracellular bacteria. cutaneous lymphocyte-associated antigen (CLA), a carbohydrate determinant defined by reactivity We investigated the mechanism by which granulysin kills a broad spectrum of microbial pathogens. with mAb HECA-452. We have shown that nearly all of the HECA-452 immunoreactivity of A predicted model generated from the NMR structure of the related protein NK lysin suggests CLA-positive human peripheral blood T cells resided on a single glycoprotein, the P-selectin that granulysin is comprised of a four alpha helical bundle motif, which is supported by glycoprotein ligand-1 (PSGL-1). As HECA-452 has been reported to bind several distinct, though spectropolarimetry analysis indicating 40%–50% alpha helical content. Synthetic peptides of related, carbohydrate structures and to identify glycoproteins of distinct molecular weight from granulysin conforming to a putative helix-loop-helix motif (aa 1–35, 36–70, 31–50) retained 50%– different leukocyte subsets, the restriction of this epitope to a single element on T cells was 80% of antibacterial activity; whereas those peptides without this predicted structure (1–20, 16– intriguing. To determine if this phenomenon was unique to T cells, we examined PSGL-1 and 35, 46–65, 61–80) had Ͻ20% activity. The structural model also predicts that the alpha helices HECA-reactive glycoproteins on other leukocyte populations believed to employ selectin-mediated are amphipathic, including 15 positively charged amino acids: 12 arginine (16%) and three lysine tissue targeting, including monocytes, neutrophils, and blood dendritic cells. Immunoblots probed residues. Chemical modification of the arginine residues caused a complete inhibition of the with HECA-452 and anti-PSGL-1 revealed 240 and 140 kDa immunoreactive bands in all three antimicrobial effects; however, modification of the lysine residues did not inhibit granulysin’s leukocyte types under standard reducing conditions. These could be reduced to a single band at antimicrobial activity. The ability of granulysin to alter bacterial membranes was measured by 140 kDa with enhanced reduction as we have reported for T cells. Moreover, material specifically incubating E. coli with colorimetric substrates for beta-galactosidase, which is normally sequestered immunoprecipitated from cell lysates by anti-PSGL-1 also showed reactivity with HECA-452, in the cytosolic space. Granulysin induced a dose-dependent increase in beta-galactosidase activity confirming that the two antibody epitopes resided on the same molecular species, rather than two suggesting leakage of enzyme from the cytosol consistent with electron microscopy which revealed distinct molecules of similar molecular weight. Tonsillar B cells did not express PSGL-1, but were that granulysin induces lesions on the surface of bacteria. These data suggest that the ability of found to express a nonreducible HECA-452 reactive species of Ͼ220 kDa. All CLA/PSGL-1 granulysin to kill microbial pathogens is dependent on interactions with the microbial cell wall or positive cell types displayed tethering, rolling, and shear resistance on both E- and P-selectin in membrane leading to increased permeability. The molecular basis of this interaction is likely physiologic shear flow in vitro. Our findings are consistent with the hypothesis that multiple mediated by the amphipathic structure of the alpha helical portions of granulysin including the leukocyte subpopulations use HECA-reactive variants of PSGL-1 protein as selectin ligands in the ionic interaction of the arginine residues with the negatively charged lipids present in the first stage of the multistep pathway of extravasation. microbial membranes. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 555

193 194 Co-Expression of E-Selectin and P-Selectin Binding Function by Native T cell Cutaneous CLAϩ T cells and Blood DC Scan Cutaneous Microvessels in Normal Skin: Evidence for Innate Lymphocyte Antigen Demonstrated in a Novel Antigen Capture Flow Assay Immunosurveillance R. Fuhlbrigge, J. Kieffer, D. Armerding and T. Kupper T. Kupper, C. Robert, R. Fuhlbrigge and U. von Andrian Harvard Skin Disease Research Center, Department of Dermatology, Brigham and Women’s Harvard Skin Disease Research Center, Boston Massachusetts Hospital, Boston, Massachusetts The accumulation of CLAϩ T cells in inflamed skin is well established but incompletely Tissue-specific leukocyte homing defines the character of immune surveillance, as well as the understood. Intravital microscopy of intact uninflamed murine ear skin allows for the objective patterns of autoimmune inflammatory disease. Targeting of T cells to skin has been associated observation and measurement of leukocyte flow through, and interactions with, skin microvasculat- with surface expression of the cutaneous lymphocyte-associated antigen (CLA) and it’s function ure. Studies performed with highly purified blood DC (uniformly CLAϩ), as well as cultured as an E-selectin ligand. We have characterized CLA, defined by binding of mAb HECA-452, as DC, showed significant tethering and rolling interactions of these cells with skin postcapillary an inducible carbohydrate epitope expressed on P-selectin glycoprotein ligand-1 (PSGL-1). venules (PCV, the site of leukocyte extravasation), but not arterioles and capillaries. Velocities of Although the correlation is strong, only inferential data has been published to date regarding the interacting DC in PCV were typically 100-fold slower than blood flow. These interactions could direct interaction of CLA with E-selectin. While the CLA carbohydrate is related to sLex, a be blocked completely with a combination of E/P selectin antibodies, and were absent in mice known E-selectin ligand, its actual structure remains unknown. Using a novel antigen capture deficient in E and P selectin. These and other data indicate that low constitutive levels of these system, we incorporated immobilized native human T cell CLA into an in vitro parallel-plate flow selectins are expressed on uninflamed skin PCV. We next applied this analysis to CLAϩ T cells. assay of selectin function. We have demonstrated specific binding of CHO cells expressing E- Naı¨ve cord blood T cells (CLA–, CD45RAϩ) showed no interactions with skin PCV under flow selectin to this native T cell CLA and its support of rolling function under physiologic shear stress conditions. In contrast, skin homing memory phenotype blood T cells (CLAϩ, CD45ROϩ) in vitro. This assay has allowed us to show, for the first time, that native T cell CLA/PSGL-1 is showed profound tethering and rolling interactions. T cell velocities in PCV were 100 fold slower both an E- and a P-selectin ligand. The behavior of native CLA was similar to that observed with than blood flow, but equal to blood flow capillaries and arterioles. These cells transiently recombinant PSGL-1 produced in CHO cells cotransfected with the 001α(1,3)-fucosyltransferase, accumulated in skin PCV via CLA/selectin interactions, but did not extravasate, and ultimately FucTVII. Both P- and E-selectin binding function, as well as HECA-452 reactivity, were resumed normal blood flow velocity upon exiting the PCV. We propose that CLAϩ T cells maintained in a truncated PSGL-1 Fc chimera containing only the terminal 19aa of mature PSGL- normally interact with constitutively expressed E and P selectin in cutaneous PCV. This ‘‘scanning’’ 1. Chimeras with additional O-glycosylation sites showed increased E, but not P, binding activity. of the endothelium has two consequences. First, CLAϩ cells are disproportionately abundant in These data, in conjunction with our previously published studies, strongly support the claim that the luminal compartment of normal skin PCV, and are thus poised to extravasate in skin that differential glycosylation of a single cell surface receptor can serve as a mechanism for regulating becomes inflamed (and thus express chemokines and CAM). Second, because CLAϩ T cells the tissue specific homing properties of memory T cells. Exploration of these structures and the spend a disproportionate amount of time in skin PCV, they are uniquely susceptible to skin mechanisms regulating their expression may lead to methods of modifying the tissue homing directed therapies (e.g. UV light). We propose that this represents innate (e.g. antigen independent) properties of circulating lymphocytes and their function in the regulation of skin disease in vivo. immunosurveillance, which is vital to cutaneous host defense.

195 196 ICAM-1 Participates in Both the Induction and Elicitation of Murine Contact Hypersensitivity Anti-CD44v10 Inhibits Alopecia Areata-Like Hair Loss in C3H/HeJ Mice Responses P. Freyschmidt-Paul, S. Seiter, M. Zo¨ller, J. Sundberg, A. Ko¨nig, R. Happle and R. Hoffmann H. Xu, D. Bullard and C. Elmets Department of Dermatology, Philipp University, Marburg, Germany; The Jackson Laboratory, Departments of Dermatology and Comparative Anatomy, University of Alabama at Bar Harbor, ME; Department of Dermatology, University of Homburg/Saar, Germany; German Birmingham, Alabama Cancer Research Centre, Heidelberg, Germany ICAM-1 (CD54) plays a key role in cell–cell interactions between T-cells and epidermal Langerhans Hair loss closely resembling human alopecia areata (AA) has been described in aging C3H/HeJ cells, keratinocytes and dermal endothelial cells. Although it is known that contact hypersensitivity mice. This hair loss can be induced in unaffected littermates by autologous transplantation of (CHS) responses are reduced in mice that have been treated with ICAM-1 antibodies in vivo and alopecic skin, thus providing an appropriate animal model for the dissection of the AA immune in ICAM-1 knockout mice, it is unclear whether the effect is on the induction and/or elicitation cascade. Recently, the splice variant v10 of the cell surface receptor CD44 (CD44v10) has been of CHS. In this study, experiments were performed to examine this issue. To assess the effect of shown to be expressed on T cells in lesional AA skin and a murine CD44v10-neutralizing antibody ICAM-1 on elicitation of the response, C57BL/6 mice were sensitized to oxazolone. Five days has been reported to impair delayed type hypersensitivity (DTH) reactions. Because AA is later, cells from these mice were adoptively transferred to mutant mice deficient in ICAM-1 characterized by a DTH-like T cell mediated immune response, we addressed the question expression or to wild type littermates. Both panels were ear challenged immediately after adoptive whether an anti-CD44v10-AB influences the onset of AA in grafted C3H/HeJ mice. After transfer. ICAM-1 knockout mice developed significantly less ear swelling than their wild type grafting alopecic skin from mice with AA onto unaffected littermates, mice were injected (i.p.) littermates (p Ͻ 0.05), indicating that ICAM-1 was important for elicitation of CHS. To evaluate twice weekly for 11 wk with either anti-CD44v10 or anti-CD44standard (anti-CD44s) or PBS the effect of ICAM-1 on induction of the response, oxazolone primed T-cells from the draining only. Immunohistochemistry of skin, lymphnodes and spleen was performed for CD4, CD8, lymph nodes of ICAM-1 deficient mice or wild type littermates were examined for production MHC-I, MHC-II, CD44s, and CD44v10 after treatment. After 11 wk PBS injected animals had of IFN-001γ and IL-4 by ELISA. Hapten primed T-cells from ICAM-1 deficient mice produced developed AA-like hair loss on 30–50% of the body. The extent of hair loss was reduced in anti- substantially less of both of these cytokines than normal control mice (p Ͻ 0.05). These findings CD44s treated mice. In contrast anti-CD44v10 treated mice did not show any hair loss. indicate that ICAM-1 murine participates in the induction of effector and regulatory T-cells in Immunohistochemistry showed a striking reduction of perifollicular CD8ϩ and to a less degree oxazolone CHS. The results provide evidence that ICAM-1 is important for both the induction of CD4ϩ lymphocytes and a decreased expression of MHC-I on hair follicle epithelium in anti- and elicitation of CHS responses in the mouse. Strategies which block the activity of this adhesion CD44v10 treated mice as compared to PBS or anti-CD44s treated animals. Our data show that molecule may therefore be useful in preventing the development of CHS and reducing the anti-CD44v10 is able to inhibit the onset AA-like hair loss in C3H/HeJ mice. At present the inflammatory response once it is established. mechanism by which this is accomplished remains unclear, but anti-CD44v10-triggered impairment of immune cell extravasation (e.g. CD8ϩ T cells), thereby decreasing their number in target tissues, might be a likely explanation.

197 198 Gene Regulation during Dendritic Cell Maturation Expression of a Membrane-Bound Chemokine, Fractalkine, and its Receptor in Skin Dendritic N. Yamada and S. Katz Cells is Regulated by Maturation Dermatology Branch, National Cancer Institute, Bethesda, Maryland E. Papadopoulos, N. Yamada, M. Saraf, D. Fitzhugh, H. Saeki, A. Blauvelt and S. Hwang Dendritic cells (DC) dramatically alter their function as they ‘‘mature’’ in vitro and in vivo. Immature Dermatology Branch, National Cancer Institute, Bethesda, Maryland DC are highly phagocytic and are highly effective at antigen processing, whereas mature cells are Cell-surface receptors such as costimulatory and adhesion molecules play important roles in the more migratory and have potent T cell-stimulatory capacity. Some of the factors involved in this association of dendritic cells (DC) and T cells and in their bi-directional cellular communication. maturation process are known, e.g. enhanced class II MHC, B7Ϫ1, B7Ϫ2, and CD40 expression. The CX3C chemokine, fractalkine, is expressed in a membrane-bound form on activated Using XS52 cells generated from newborn epidermis of BALB/C mice (J Immunol 154:2697, endothelial cells and mediates attachment and firm adhesion of T cells. We have shown that 1995), we have established culture conditions (GM-CSF and IL-4 for 6 d followed by TNF- fractalkine is associated with Langerhans cells in human epidermis. To determine whether 001α, IL-1001β, and agonistic anti-CD40 mAb for 3 d) that cause these immature cells to become fractalkine is involved in DC–T cell interactions, we sought to characterize the regulation of fully mature (XS-DC) as evidenced by morphology, phenotype, and potent allostimulatory fractalkine expression in both Langerhans cells and model DC. Epidermis derived from suction capacity. This model system enabled us to identify genes that are associated with the maturation blister roofs from healthy human volunteers was placed into culture for 2–3 d and mature of DC using cDNA microarray analysis, followed by quantitation through phosphorimaging. In migratory DC harvested. For comparison, epidermal Langerhans cells were isolated from fresh combination with RT-PCR, more than 700 genes have been screened and positive results were epidermal suspensions using HLA-DR antibodies and immunomagnetic beads. Human peripheral verified by RNase protection assays. Neoexpressed genes in XS-DC not expressed in XS52 were blood monocytes were treated with IL-4, GM-CSF, and TGF-001β for 1 wk to obtain immature IL-1001β, RANTES, ABCD-1, Fractalkine, and CCR7. Those upregulated (Ͼ3x greater than DC and further stimulated with monocyte conditioned medium for2dtostimulate maturation. XS52) were class II, class II transactivator (CIITA), CD40, CD44, TNF001α, LT001β, M-CSF, The expression of fractalkine was studied by RT-PCR and flow cytometric analysis using an IL-1RA, Shc, Butyrate Response Factor-1, Rel-B, and I001κB001α. Downregulated genes (Ͻ1/ antifractalkine specific antibody. In another model of DC maturation, fractalkine expression was 3x as compared to XS52) included CD14, MIP-001α, MIP-1001β, MIF, Neuroleukin, IL-1R studied in the murine immature DC cell line, XS52, which can be induced to mature with type II, Plasminogen Activator Inhibitor-2, PKC001θ, Nm23-M2, Rab-2, and Bax. NGF001α cytokines or anti-CD40 ligation. Langerhans cells could be efficiently isolated from epidermal and TGF001β1 were maximally expressed by IL-4-treated XS52 and downregulated in XS-DC. suspensions. Mature, migratory Langerhans cells showed increased expression of fractalkine mRNA Many of the identified genes are associated with critical cellular metabolic properties. These gene in comparison with freshly isolated, resting Langerhans cells, which displayed weak expression. expression findings were corroborated in studies of immature and mature C3H-3B7 cells (an Peripheral blood-derived dendritic cells showed up-regulation of fractalkine mRNA upon immature DC line derived from fetal skin that has even greater similarity to Langerhans cells than maturation. Cytokine-induced maturation of XS52 cells resulted in the up-regulation of fractalkine XS52). Taken together, our studies demonstrate the utility of using cytokine-manipulated cells to and, interestingly, down-regulation of its receptor, CX3CR1, as determined by RNAase protection. study molecular-phenotypic-functional correlations in DC. In conclusion, the expression of fractalkine is enhanced during the process of DC maturation. By analogy to CX3CR1-mediated adhesion of T cells to activated endothelial cells, the enhanced expression of fractalkine during maturation may play a role in the ability of T cells and mature DC to form conjugates and engage in cell–cell communication. 556 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

199 200 Langerhans Cell-like Dendritic Cells (DC) from both Susceptible and Resistant Mice Release IL- MHC Class I-Binding and Immunostimulatory Properties of Peptides Derived from T Cell 12 p70 after Infection with Leishmania major Receptors of CTCL Cells E. von Stebut and M. Udey D. Winter, P. Meraner, C. Brna, R. Strohal, F. Trautinger, R. Knobler, G. Stingl and D. Maurer Dermatology Branch, NCI, NIH, Bethesda, Maryland DIAID and Division of Env. Dermatology, Department of Dermatology, University of Vienna Leishmania major amastigote-infected C57BL/6 (B6) fetal skin-derived DC (FSDDC), unlike M∆, Medical School, Vienna, Austria are activated and release cytokines (including IL-12 p70) and likely initiate protective Th1 A candidate tumor specific-antigen in cutaneous T cell lymphoma (CTCL) is the clonotypic T immunity (J Exp Med 188:1547). To characterize differences in DC function in mice that are cell receptor (TCR). However, limited information exists concerning the class I-binding ability genetically susceptible (BALB/C) and resistant (B6) to leishmaniasis, we analyzed the effects of L. and, more importantly, the immunogenicity of human TCR-derived peptides. In order to generate major on FSDDC from both strains. BALB/C- and B6-DC ingested similar numbers of parasites. essential tools to address these issues, we cloned and sequenced cDNAs encoding the idiotypic ϩ Like B6-DC, infection of BALB/C-DC led to upregulation of MHC I and II, CD40, CD54, TCR001α and 001β chains of CD4 CTCL cells. Using anti-TCRV001β-specific mAbs, CD80 and CD86 within 18 h. L. major-induced BALB/c-DC activation also led to the release of expanded CTCL cells were identified and isolated by flow cytometry. Selective TCRV001α/β TNF001α, IL-6 and IL-12 p40 (483 001Ϯ 87 pg per 2 001ϫ 105 DC, n ϭ 8) into 18 h usage of sorted cells was confirmed by a V region-specific PCR assay and the clonality of TCR supernatants. Interestingly, infected BALB/C-DC and B6-DC both released IL-12 (p70) into 72 amplicons was assessed by DNA sequence comparisons of multiple cDNA subclones. We have α α α β h supernatants (35 001Ϯ 28 vs 4 001Ϯ 2 pg per 106 DC, n ϭ 6). Additional stimulation with isolated the cDNAs encoding the entire clonal TCR (V001 21-N-J001 50-C001 /V001 17- γ N-D001β1.1-J001β1.4-C001β1 and V001α6-N-J001α29-C001α/V001β17-N-D001β-J001β2.7- IFN001 , anti-CD40 or both induced the release of more p70 from infected BALB/C-DC (123 β β β β β 001Ϯ 78, 116 001Ϯ 53, and 244 001Ϯ 103 pg per 106 DC, n ϭ 6) than B6-DC (25 001Ϯ 7, C001 2] in two cases and the clonal TCR001 -encoding cDNA (V001 6-N-D001 -J001 1.5- C001β1) in an third patient. The deduced TCR amino acid sequences from the first patient 23 001Ϯ 8, and 52 001Ϯ 8 pg per 106 DC, n ϭ 8). Thus, skin DC from genetically susceptible (HLA-A2ϩ) were subjected to an anchor position-based motif search for nonameric HLA-A2 and resistant mice were activated by L. major with upregulation of MHC Ag and costimulatory peptide ligands. Peptides located within the V-, CDR3-, and C-regions from either TCR subunit molecules. Infection also led to release of cytokines (including IL-12 p70) from DC of both were selected based on their T1/2 score of Ͼ10. Binding of these peptides was tested on HLA- mouse strains. Genetic susceptibility to L. major that results from induction of Th2 predominant A2ϩ TAP-deficient T2 cells. Following acid-mediated disassembly of surface-bound MHC class immune responses does not appear to reflect a difference in the ability of DC/Langerhans cells to I/peptide/001β2 m complexes, cells were exposed to TCR-derived peptides in the presence of internalize or respond to parasites. Susceptibility could reflect a defect in recruitment of DC in exogenous 001β2 m and the ability of peptides to reconstitute conformationally intact HLA-A2 the initiation phase of the immune response or result from an abnormality not manifested in DC. was monitored using mAb W6/32. Peptides VIFGPTSL (CDR3001α) and ILWLQPDWV (V001α21) reconstituted class I complexes in a magnitude comparable to that achieved by a high- avidity HLA-A2 binding, hepatitis B virus-derived control peptide (Kd ϭ 3.3 001ϫ 10–9 M). In binding competition assays, the HBV control and the V001α21 peptides displayed similar affinities for HLA-A2 while the CDR3001α peptide bound HLA-A2 even more efficiently than did the HBV peptide. The four remaining TCR (V001β17, C001β1, CDR3001β, C001α) peptides also reconstituted W6/32 immuno-reactivity and competed with HBV peptide binding although less eficiently than the V001α21- and CDR3001α-derived moieties. To see whether TCR-derived peptides are presented in immunologically relevant fashion and, furthermore, can sensitize target cells for MHC class I-restricted cytotoxicity, T lymphocytes from HLA-A2ϩ donors were repeatedly stimulated with V001α21 peptide-loaded dendritic cells. T cell lines thus generated were tested for cytotoxicity against peptide-pulsed T2 cells. T cell blasts efficiently lysed V001α21 but not gp100/pmel17 control peptide-loaded targets. Immunodepletion experiments further revealed that this peptide-specific cytotoxic activity is mediated by CD8ϩ rather than by CD4ϩ T cells. Thus, it appears that clonotypic TCRs of CTCL cells can contain several high-affinity MHC class I-binding peptides and that at least for certain epitopes peptide-specific CTL precursors exist. Provided that the proteasomal machinery of CTCL clones allows the generation and presention of TCR-derived peptides in vivo, some of these moieties may well serve as relevant immunogens for vaccination strategies aiming at inducing protective antitumor immunity.

201 202 HLA-DR1 His81beta Residue Influences the Response of a Peptide-Sequence Independent Cellular Events in Experimental Bullous Pemphigoid: Mast Cells, but not T or B Lymphocytes, Nickel-Reactive Human T Cell Clone Natural Killer Cells, or Macrophages, Play a Major Role in the Early Stage of Subepidermal Blis- C. Moulon, J. Vollmer, D. R. Karp, J. van Bergen, F. Koning, D. Wild and H. U. Weltzien tering Max-Planck-Institute for Immunbiology, Freiburg, Germany; UT South-western Medical Center, Z. Liu, X. Zhou, M. Fleming, G. Giudice and L. A. Diaz Dallas, Texas; Leiden University Medical Center, Leiden, the Netherlands Department of Dermatology, Medical College of Wisconsin, and VA Medical Center, Mil- The antigenic epitopes recognized by Ni-specific human CD4ϩ and CD8ϩ T cells remain so far waukee, Wisconsin unidentified. Different models are often proposed to explain metal-specific activation of T cells: Bullous pemphigoid (BP) is an autoimmune disease characterized by subepidermal blisters and (i) Metal-specific TCR are activated through contacts with metal-modified amino acid residues autoantibodies against two hemidesmosomal proteins – BP230 and BP180. The subepidermal of self peptides embedded in the MHC binding groove; (ii) Metal-specific TCR recognize metal- blisters in BP are associated with an inflammatory cellular infiltrate composed of neutrophils, modified amino acid residues of the MHC molecule itself, or a change of the MHC conformation eosinophils, lymphocytes, monocyte/macrophages and mast cells. We have shown that passive induced by metal coordination; (iii) Metals interfere with the processing of self antigens and this transfer of anti-BP180 IgG into neonatal mice induces subepidermal blisters that are dependent results in the induction of T cells recognizing cryptic antigens. We have recently found that TCR ϩ on C activation and neutrophil infiltration. In this study we investigated the role of these VB17 elements are over-represented among TCR of T cell clones from highly nickel-allergic inflammatory cells in experimental BP. Pathogenic anti-BP180 IgG induced extensive subepidermal donors. In order to gain insight in the mechanism of activation of these T cells, we have ϩ ϩ blisters in scid mice lacking both B and T lymphocytes and in 2-microglobulin (2 m)-deficient characterized a nickel-reactive VB17 CD4 human T cell clone and a mouse transfectant mice lacking natural killer cells (NK). BALB/C mice pretreated with carrageen to deplete expressing the TCR originating from this clone: Nickel-specific T cell proliferation and/or IL2 macrophages did not develop skin lesions after injection with pathogenic IgG. Mast cell-deficient production can be inhibited by anti-HLA-DR antibodies. Homozygous B cells of different mice were also resistant to experimental BP. Myeloperoxidase MPO activity levels in the skin of haplotypes can present Ni, indicating that the TCR is HLA-DR-promiscuous. Glutaraldehyde- fixed APC are able to activate the T cells in the presence of Ni, thereby excluding the necessity the anti-BP180 injected animals were used as a measure of neutrophil recruitment. Compared for Ni to enter in the cells. HLA-DR1-transfected 293 cells, supertransfected with a Ii vector with MPO levels in BALB/C mice, mast cell deficient mice showed a 96% reduction and encoding different T helper epitopes, are very efficient APC for the clone and the transfectant, as carrageen-treated mice showed a 27% reduction in MPO activity. Scid mice and 2m-deficient compared to HLA-DR1 homozygous B cells or HLA-DR1 transfected DAP cells. This suggests mice showed MPO levels only slightly below those of controls. Finally, mast cell-deficient mice that T cell activation can be induced by 293 APC expressing MHC molecules mainly loaded pretreated with the proinflammatory cytokines, TNF-, IL-1, IL-6, or IL-8, became susceptible to with a single peptide, different for each APC. In addition, DAP cells expressing the wild-type pathogenic anti-BP180 IgG. These findings suggest that mast cells play a major role in subepidermal DR alpha chain, in association with mutant HLA-DR1 beta chains (His81 mutated into Ala, Asp blistering in experimental BP and macrophages might play an accessory role. T and B lymphocytes or Glu) are not able to induce nickel-specific activation, while they have retained capacity to and NK cells do not appear to play any role in this disease process. These findings suggest that induce SEB-specific activation. We propose an additional model of Ni-specific T cell activation, blister formation in BP is triggered by certain inflammatory cells and their released mediators. independent of processing and independent of the peptide sequence loaded on MHC class II molecules. Our data also suggest that the residue His81 in the beta chain of the HLA-DR1 molecule influences the response of nickel-specific CD4ϩ T cells and are currently investigating this mechanism of activation.

203 204 Overexpression of the Endogenous Angiogenesis Inhibitor TSP-1 Inhibits Tumor Growth and Blocking TGF01β Signaling In Transgenic Epidermis Accelerates Chemical Carcinogenesis via Angiogenesis in Human Squamous Carcinoma Cells Aberrant Cell Cycle Progression and Angiogenesis M. Streit, P. Velasco, L. F. Brown, M. Skobe, L. Richards, J. Lawler and M. Detmar C. Go,* Ping Li and Xiao-Jing Wang Cutaneous Biology Research Center/Department of Dermatology, Massachusetts General Hospital Departments of Cell Biology, Dermatology, *Otolaryngology, Baylor College of Medicine, and Harvard Medical School, Charlestown, Massachusetts Houston, Texas The generation of new blood vessels is essential for carcinomas to grow beyond minimal size. We Mutations in the transforming growth factor 01β type II receptor (TGF01βRII) have been recently found that the endogenous angiogenesis inhibitor, thrombospondin-1 (TSP-1), was identified in human cancers, suggesting a causal role for loss of TGF01βRII in cancer development. downregulated during epidermal skin carcino-genesis. To further characterize the function of To directly test this in vivo, we have generated transgenic mice expressing a dominant negative TSP-1, we investigated tumor growth and angiogenesis of TSP-1 overexpressing A431 and SCC- TGF01βRII (01∆βRII) in the epidermis, by utilizing a truncated mouse loricrin promoter (ML). 13 cutaneous carcinoma cells, growing as intradermal xenotransplants in nude mice. Enhanced When these mice were subjected to a standard two-stage chemical carcinogenesis protocol, they TSP-1 mRNA expression and protein secretion were confirmed in stably transfected tumor cell exhibited an increased susceptibility compared to nontransgenic mice, with greatly accelerated clones by Northern and Western blots, and biological activity of transfected TSP-1 was confirmed benign papilloma formation, malignant conversion and metastasis. Flow cytometry of ML.01∆βRII by the antiproliferative effect of conditioned media on human dermal microvascular endothelial tumor cells showed a significant reduction in the G1 population and a 6-fold higher G2/M cells. Although tumor cell proliferation in vitro was unaffected, in vivo tumor growth was potently population. Consistent with the perturbed cell cycle, ML.01∆βRII tumors also showed loss of reduced in TSP-1 overexpressing A431 and SCC-13 xenotransplants as compared to control expression of p21 (WAF-1) and p15 (INK4b), inhibitors of cyclin-dependent kinases (cdks). tumors. TSP-1 overexpressing tumors were characterized by large areas of necrosis and by Furthermore, ML.01∆βRII tumors exhibited increased angiogenesis and progressed to metastases decreased microvascular density, as analyzed by morphometric evaluation of vessel numbers in even though the primary tumors were still classified as well-differentiated carcinomas. Increased CD31 immunostains. The average blood vessel area was reduced by 50%–75% in TSP-1 expression of vascular endothelial growth factor (VEGF), an angiogenesis factor, and decreased overexpressing tumors, with complete inhibition of blood vessels of more than 2000 µm2 area expression of thrompsondin-1 (TSP-1), an angiogenesis inhibitor, were also observed in size. These results suggest an important role of TSP-1 for the control of epidermal skin ∆β β carcinogenesis and epithelial skin cancer growth. ML.01 RII tumors. These data provide in vivo evidence that inactivation of TGF01 RII accelerates skin tumorigenesis by loss of cell cycle control and increased angiogenesis. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 557

205 206 E6/E7 mice – A Model for Human Papillomavirus-Dependent Disease Reduced UV-Induced Carcinogenesis in Mice with a Functional Disruption in B7-Mediated J. Crish, F. Bone, S. Balasubramanian, T. Zaim, T. Wagner, J. Yun, E. Rorke and R. Eckert Costimulation S. Beissert, J. A. Bluestone,* I. Mindt, M. Voskort, D. Metze, A. Mehling, T. A. Luger, T. Physiology & Biophysics, Dermatology, Biochemistry and Oncology, Case Western Reserve Schwarz and S. Grabbe University, Cleveland, Ohio; and Edison BioTechnology Center, University of Ohio, Athens, Ohio Department of Dermatology, University of Mu¨nster, Mu¨nster, Germany; *Ben May Institute for Human papillomavirus is an important etiological agent in a variety of surface epithelial cancers. Cancer Research, The University of Chicago, Chicago, Illinois An important problem has been the lack of an appropriate in vivo, model for HIPV-dependent Immunosuppression by UV irradiation (UVR) regulates the induction of skin cancer by suppressing human cancer. Some models presently exist, but these all appear to have deficiencies. To develop cell-mediated immune responses. The costimulatory molecules B7-1 and B7-2 bind to their an appropriate model, we used the involucrin promoter to drive expression of the human counterreceptors CD28 and CTLA-4 and provide costimulatory signals essential for antigen- papillomavirus type 16 E6/E7 oncoproteins in the superficial layers of surface epithelia in mice. specific T cell activation. To investigate the role of the B7/CD28-CTLA-4 signaling pathway in A key feature of this model is that the pattern of HIPV oncoprotein expression in the E6/E7 photocarcinogenesis, we utilized transgenic (Tg) mice which express CTLA-4Ig, a high affinity mouse epithelia is very similar to that observed in human disease (i.e. expression is observed in CD28/CTLA-4 antagonist that binds to B7-1 and B7-2. The transgene is driven by a skin-specific the suprabasal layers). In fact, in every way measured, both biochemical and morphological, this promoter yielding high levels of CTLA-4Ig in the skin and serum. Groups of Tg and controls model mimics the human disease. These mice display extensive hyperplasia with cell division (both n ϭ 20) were irradiated with UVB (10 kJ per m2) 301ϫ a week for 6 mo. Chronic UVB confined to the suprabasal layers. In spite of this hyperplasia, no tumors are, produced. These exposure of CTLA-4Ig Tg mice resulted in significantly fewer skin tumors (eight tumors on seven features, hyperplasia with suprabasal cell division without tumor. formation, are hallmarks of early mice), when compared to control mice (21 on 16). Moreover, the probability of tumor HPV-dependent human disease. Treatment with carcinogen can advance the animals to form development was significantly reduced in Tg compared to control mice (p Ͻ 0.002). To investigate papillomas, a finding that fits with the concept that these animals are predisposed towards cancer whether Tg mice develop systemic immunosuppression after UVR, we investigated delayed-type provided the appropriate secondary oncogene is activated. HPV E6/E7 transcript splicing is hypersensitivity (DTH) responses to alloantigen in UV-exposed Tg and control mice. Mice were identical to that observed in human disease. Finally, tumor suppressor protein expression and cell UV irradiated, sensitized with allogeneic spleen cells and then challenged with alloantigen at one cycle regulatory protein expression is altered in a manner that is consistent with observations in hind footpad and footpad swelling was assessed. Interestingly, Tg mice were resistant to UV- HPV-positive human tissues (i.e. increased levels of the p53, p21, p27, cdk2, cdk4, cdk6, cyclin induced suppression of DTH responses to alloantigen. Since it is still unclear whether UV-induced D1, and cyclin E regulatory proteins). The altered pattern of cell cycle regulatory protein expression immunosuppression is caused by regulatory/suppressor T cells or by a shift from Th1 to Th2 we is consistent with the hypothesis that the epidermal, phenotype is primarily driven by the analysed the production of IL-4 and IFN-01γ by Tg and control T cells. Mitogen-stimulated E7 oncoprotein. CTLA-4Ig Tg T cells produced significantly less IL-4 but higher IFN-01γ levels as compared to control T cells, suggesting an impaired Th2 response and a relative increase of Th1-type immunity. Together, these data provide strong evidence that B7 engagement directs immune responses towards the Th2 pathway and that the generation of Th1 immune reactions is crucial for the immunological protection against photocarcinogenesis. Furthermore, they support the concept that UVR suppresses the immune system by inducing a shift from a Th1 to a Th2 response.

207 208 Thymidine Dinucleotide Pre-Treatment Reduces DNA Mutation Frequency Generation and Characterization of Dendritic Cell-Tumor Hybrid Clones M. Khlgatian, I. Hadshiew, M. Eller, H. Giese,* J. Vijg* and B. Gilchrest H. Matsue, K. Matsue, A. Morita and A. Takashima Boston University School of Medicine, Boston, Massachusetts; and *Harvard Medical School, Department of Dermatology, UT South-western Medical Center, Dallas, Texas Boston, Massachusetts Anti-tumor immunity has been induced experimentally by inoculation of dendritic cells (DC) It was previously shown that treatment of mammalian cells with the small DNA fragment that are loaded with relevant tumor-associated antigens (TAA) in a form of whole cell lysates, thymidine dinucleotide (pTpT) induces protective responses including increased melanogenesis proteins, peptides, RNA or DNA. A new TAA-loading strategy was introduced recently, in which and enhanced capacity to repair DNA damaged by UV or chemical carcinogens. This is DC were physically fused with tumor cells. Although those DC-tumor cell hybrid preparations accomplished largely through the activation of p53 and subsequent induction of p53-regulated (containing relatively large numbers of nonfused cells) induced protective immunity, this technology genes. Because increased DNA repair is associated with decreased mutation rate in human cells, requires further refinement of the methods to select DC-tumor hybrids. In this study, we developed we used transgenic mice carrying multiple genomic copies of a LacZ reporter plasmid to examine a unique ‘‘double-selection’’ strategy by using the long-term DC line XS106 (derived from A/J the effect of pTpT pretreatment on UV-induced mutations. Embryonic mouse fibroblasts were mice) and examined the clonal heterogeneity of resulting hybrids. We first selected the HAT- treated with 100 µM pTpT or an equal volume of diluent for 3 d prior to sham irradiation or 2 sensitive variant (termed XS106–7) by culturing XS106 cells with 8-azaguanine and then introduced mJ per cm2 or 5 mJ per cm2 UVB (solar-simulated, metered at 285 Ϯ 5 nm). pTpT treatments a zeocin resistance gene. The resulting DC clone (termed XS106–7 Zeo) was found to be highly and irradiations were repeated after 2 wk. The plasmids were recovered 1 wk later from genomic sensitive to HAT and resistant to zeocin, while maintaining all the immunological properties of DNA by restriction enzyme digestion and specific binding to the LacI protein. Mutant LacZ the original XS106 DC line. When these DC were fused, using a standard PEG protocol, with a plasmids were positively selected by transfection into bacteria and growth on selective medium. GFP-transfected subclone (S1509a-GFP) of the S1509a fibrosarcoma line (derived from A/J mice), The mutation frequency, defined as the number of mutant plasmids per 105 total plasmids, in only 0.3%–1% of the cells expressed both CD45 (derived from DC) and GFP (derived from diluent- versus pTpT-treated cells was the same in cells not irradiated or sham-irradiated with tumor cells). In theory, those rare DC-tumor hybrid cells should be able to survive in the double- either dose (14 Ϯ 1 vs 17.8 Ϯ 9, 19.7 Ϯ 2 vs 16.6 Ϯ 4, and 17.2 Ϯ 4 vs 15.8 Ϯ 9, respectively). selection medium containing HAT and zeocin, whereas nonfused DC and nonfused tumor cells As expected, UV exposure increased mutation frequency and following UV doses of 2 mJ per should be eliminated by HAT and zeocin, respectively. In fact, all of the 55 hybrid clones cm2 and 5 mJ per cm2, the mutation rate in pTpT-treated cells was 45% to 56% lower than in established by limiting dilution in the double-selection medium expressed both CD45 and GFP. control, diluent-treated cells (33.6 Ϯ 3 vs 61.5 Ϯ 9 and 47.6 Ϯ 3 vs 108.5 Ϯ 54, respectively). Importantly, we observed significant clone-to-clone heterogeneity in terms of surface phenotype In order to determine the effect of pTpT treatment in vivo, mice had 100 µM pTpT in propylene and function. Although all clones expressed MHC class I molecules, only two clones expressed glycol applied to one ear and vehicle alone to the other ear daily for 4 d and on day 5 both ears MHC class II molecules and 16 clones expressed CD86. Some clones lost their proliferative were exposed to 100 mJ per cm2 UVB light. This protocol was repeated weekly for 3, 5, or 7 responsiveness to GM-CSF [3H-thymidine uptake], the potential to secrete IL-101β in response wk (three mice per group). The nonpigmented mouse ears did not tan during the experiment. to LPS stimulation [ELISA], and/or the capacity to activate naive T cells [primary allo-MLR]. In One week after the final irradiation, LacZ plasmids were harvested from the ear epidermis and sum, only two clones (S6 and S11) were judged as bona fide DC-tumor hybrids. A single s.c. the mutation frequency was determined. After 3, 5, and 7 wk, pTpT-treated skin exhibited a injection of the hybrid clone S6 (2 01ϫ 105 cells per mouse) inhibited the growth of subsequently 20%–30% lower mutation frequency than diluent treated skin (200 vs 293, 155 vs 216, and 261 inoculated S1509a tumor cells in A/J mice, documenting that an appropriately selected hybrid vs 322, respectively). This represents slightly less protection than observed in vitro and is consistent clone can effectively initiate protective immunity. We propose that the present study forms a with less pTpT delivery through skin than in medium. The data show that pTpT-enhanced DNA technical platform for the clinical application of the DC-tumor hybrid therapy. repair reduces UV-induced mutations in vitro and in vivo and suggest that topical application of this dinucleotide could be used to lower the mutation rate in carcinogen-exposed human skin.

209 210 Early Detection of Melanoma by Tumor Cell Labeling with Green Fluorescent Protein Using Proteomics to Analyze the Signaling Status of Melanoma Cell Lines H. Yang, T. Galloway, J. Chang, J. Arbiser, J. C. Ansel and C. A. Armstrong K. Resing, J. Yeh, N. Nguyen, D. Marr, M. Fujita, N. Ahn and D. Norris Department of Dermatology, Emory University, Atlanta, Georgia Department of Chemisttry and Biochemisry, University of Colorado, Boulder, Colorado; Depart- The early stages of tumor progression and micrometastasis are difficult to detect due to the inability ment of Dermatology, University of Colorado Health Science Center, Denver, Colorado to identify tumor cells among normal host cells. However, utilization of green fluorescence protein Disease progression in malignant melanoma involves distinct stages of invasive growth followed (GFP) as a tumor cell maker could allow early tumor micrometastases to be visualized more by metastasis, resulting in complex changes in cell signaling that are difficult to identify. Methods accurately. This will be particularly valuable for assessing the response to different therapies in an to monitor all of the signaling changes would increase our understanding of cancer etiology and experimental animal model of cutaneous melanoma. We have established stable, GFP-producing facilitate diagnosis and treatment. We are using a powerful new technique (proteome analysis) to murine melanoma cell lines by retroviral transduction. After FACS, more than 90% of infected detect changes in protein expression and post-translational modification in melanoma cell lines. cells expressed GFP. These GFP-producing cell lines were utilized in syngeneic mice to investigate Because changes are directed by transcriptional regulation, message stability, and/or activation of the process of tumor metastasis and progression in distant organs. Micrometastases could be modulating enzymes, the proteome reflects the cell’s signaling status. Proteins in cell extracts are detected and visualized by GFP fluorescence at the single-cell level in lung, liver and brain tissues resolved by 2D PAGE to detect changes in intensity, size, or charge, then identified by excising at day 15, 15 and 32, respectively, following the subcutaneous injection of 1 01ϫ 106 B16-GFP gel ‘‘spots’’, proteolyzing in-gel, and extracting peptides for analysis by mass spectrometry in order or HFH18-GFP melanoma cells. In contrast, no metastases were detected by routine histologic to identify the protein by data base searching, or to design oligonucleotide primers for cloning. staining methods in any mice following subcutaneous injection of B16 or HFH18 melanoma cells. We have compared cell lines derived from radial growth phase melanoma (WM35) or advanced The respective GFP-producing tumors grew to the same average tumor volume over the 32 day invasive, metastatic melanoma with WM35 derived lines stably transfected with constitutively study period as the B16 and HFH18 parental cells. These findings are the first demonstration of active Ras or dominant negative p53. This study identified several proteins expressed in invasive, any metastases following subcutaneous injection of HFH18 melanoma cells and of brain metastasis metastatic melanoma, that were not present in the noninvasive WM35 or the transfected cell following subcutaneous injection of any murine melanoma cell line. Thus, GFP melanoma cell lines. It also provided a wider view of cell regulation by Ras and p53, demonstrating the usefulness transduction provides us with a new, sensitive, and powerful tool for the early detection of tumor of proteome studies for functional analysis of selected activated or inhibitory genes in melanoma. cell dissemination and progression and may greatly expand the utility of murine melanoma model systems for developing novel therapies for cutaneous melanoma. 558 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

211 212 Genomic Analysis of Human Squamous Cell Carcinoma Gene Expression Via cDNA Microarrays The Insulin-Like Growth Factor Receptor Promotes the Survival of Human Keratinocytes H. Fan, C. Barry, Q. Lin, S. Baek, P. Brown and P. Khavari Following Ultraviolet B Irradiation at the Expense of Cellular Proliferation VA Palo Alto and Stanford University, Stanford, California C. Kuhn, M. Kumar, J. Cotton, S. Hurwitz and D. Spandau Abnormal epithelial growth is a hallmark of squamous cell carcinoma (SCC), however, the changes Departments of Dermatology, Pathology and Biochemistry, Indiana University School of Medicine, in gene expression that underlie this process are not well understood. To study this issue, we Indianapolis, Illinois sought to determine the gene expression differences between invasive SCC and normal skin at a The response of human keratinocytes to ultraviolet B (UVB) radiation is directly dependent on genomic scale. mRNA was prepared from lesional SCC tissue and site matched normal control their nutritional status. We have previously reported that if keratinocytes are deprived of either skin from the same patient. mRNA was then reverse transcribed using fluorescent nucleotides insulin or insulin-like growth factor-1 (IGF-1), they become more sensitive to UVB-induced and hybridized to a total of approximately 12,500 genes via high density cDNA microarrays. A apoptosis. This sensitivity of keratinocytes to UVB-induced apoptosis was mediated exclusively total of 94 genes whose expression was either dramatically enhanced [81 genes] or reduced [13 through the IGF-1 receptor. In the presence of IGF-1 receptor activation, keratinocytes were able genes] in SCC versus normal skin were identified for further study, including genes involved in to survive UVB irradiation, but as a consequence to survival, the keratinocytes lost the potential cell cycle promotion, signal transduction, matrix degradation, differentiation and inflammation as to continue cellular replication. Inhibition of the IGF-1 receptor resulted in a higher proportion well as ESTs of unknown function. Following confirmatory analysis using northern blotting, of apoptotic cells following UVB irradiation, but the keratinocytes that survived maintained the differentially expressed genes were then used to probe an additional panel of SCC lesional/site ability to proliferate. Therefore, the role of the IGF-1 receptor in response to UVB radiation may matched normal skin mRNA specimens (n ϭ 6 separate patients) to confirm reproducibility of be to suppress the ability of keratinocytes with UVB-damaged DNA to replicate, while maintaining differential expression. Specific genes were found to be either induced or repressed in a majority the barrier function of the epidermis (i.e. repress the loss of epidermal cellularity due to apoptosis). of SCC specimens compared to normal site matched skin from the same individual. Included We have begun to further define the signal transduction pathways required for the survival among genes strongly upregulated in SCC are P-cadherin, lipocalin and stromelysin-1, with the function provided by the IGF-1 receptor. Both phosphatidylinositol 3-kinase (PI3K) and MAP latter expressed 81.4-fold higher in SCC than in normal skin. Among the genes strongly repressed and ERK kinase 1 (MEK1) play a role in transducing the survival signal from the IGF-1 receptor. in SCC are the tumor suppressor EGR-1 and the Prostaglandin E receptor, EP4 subtype. Of We also provide evidence that the loss of the proliferative potential of human keratinocytes interest, the six individual SCCs studied appeared to fall into two different groups based on the following UVB irradiation is due to the induction of premature senescence. expression patterns of the studied genes, suggesting that SCCs may comprise distinct subtypes at the molecular level. These data identify specific patterns of induced and repressed genes in human SCC and form a basis for future efforts focused on understanding basic mechanisms of epithelial growth control as well as the pathogenesis, prognosis and therapy of this cancer.

213 214 Mutation Analysis of Basal Cell Carcinoma Margins Examining the Role of p73, a p53 Homolog, in Skin Carcinogenesis S. Kanjilal, B. R. Nelson, N. Banerji and V. Kapur K. Liefer, X. Wang, A. Yang, F. McKeon and D. Roop University of Minnesota, Twin Cities, Minneapolis; and University of Texas, Houston, Texas Departments of Cell Biology and Dermatology, Baylor College of Medicine, Houston, Texas; and Basal cell carcinomas (BCC) of the skin are the most common human cancers with more than Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 800,000 new cases reported each year in the USA. Although these tumors rarely metastasize, they The protein product of the p53 gene is a classical tumor suppressor which is mutated in greater are often locally invasive and have ill-defined margins. Retrospective analysis of reports published than half of all human tumors, including skin cancer. The exact role of p53 in skin carcinogenesis, during the last 50 y indicates that nearly 10% of primary BCC (previously untreated) and 15%– however, has been difficult to elucidate. Previous skin chemical carcinogenesis experiments using 40% of previously treated cases recur within five years after treatment. The recurrence rates are mice null for p53 revealed that these mice rarely developed skin tumors before succumbing to reduced to 1% and 6%, respectively, when primary or recurrent BCCs are excised by the Mohs’ internal tumors at a rate similar to untreated p53 null mice, implying that the skin is more resistant technique wherein deep and peripheral margins are verified by microscopic examination to be to tumorigenesis resulting from loss of p53 than other organs. This suggests a potential back up free of malignant cells. In previous studies, we have shown that alterations in DNA (e.g. p53 mechanism in the skin that compensates for loss of p53. Conversely, mice expressing a mutant mutations and LOH at H-ras) are often present in the apparently normal tissue remaining after form of p53 in the epidermis exhibited an increased susceptibility to skin chemical carcinogenesis resection of nonmelanoma skin cancers by non-Mohs’ modalities. Here we report our analysis of suggesting that this dominant negative p53 not only interferes with p53 function but perhaps with matched sets of BCC and tumor-adjacent normal tissue obtained from 20 patients treated by this potential compensatory mechanism. The recent identification and cloning of p73, a p53 Mohs’ surgery. Consistent with our expectations, the tumor-adjacent tissues were mutation-free homolog, which shares sequence and functional similarities to p53 and has been shown to be in 19 (95%) of these cases. Since mutations in the margins may predispose towards tumor expressed in the skin, makes this an interesting candidate for the compensatory mechanism in the recurrence, we have attempted to develop a powerful method for the rapid analysis of mutations skin. We have used mice null and heterozygous for p73 in the skin chemical carcinogenesis in tissue sections. Preliminary studies suggest that the use of a novel semiautomated sequence protocol. Preliminary data suggests that these mice respond quite similarly to wild-type littermates analysis method coupled with a rapid polymerase chain reaction (PCR) amplification protocol with respect to kinetics of papilloma formation; however, these papillomas are larger and appear will enable the detection of mutations in clinical samples within 2 h after collection. The overall to convert to carcinoma more quickly than wild type controls. goal of this project is to make DNA diagnostics available for the real-time analysis of mutations during resection of highly aggressive tumors by the Mohs’ technique.

215 216 Ultraviolet Radiation Inhibits the Expression of CUSP, a Member of the p53 Family The Full-Length Calcium Sensing Receptor (CaR) is Required for Normal Epidermal Differen- A. Marchbank, R. Dellavalle, T. Egbert, J. DeGregori, L.- J. Su, P. Walsh and L. A. Lee tiation University of Colorado School of Medicine; Denver Health Medical Center and Department of C. -L. Tu, Y. Oda, L. Komuves, C. Ho and D. D. Bikle Veterans Affairs, Denver, Colorado Endocrine Unit and Department of Dermatology, VA Medical Center and University of The carcinogenic importance of p53, the prototypical tumor suppressor gene, is highlighted by California, San Francisco, California and Department of Genetics, Harvard Medical School, the finding that p53 is mutated in more than half of human tumors. DNA damage induces p53 Boston, Massachusetts expression which arrests the cell cycle, allowing DNA repair prior to mitosis, or signals We have recently discovered that the calcium sensing receptor (CaR) is expressed in human overwhelmingly damaged cells to undergo apoptosis. Recently, two homologues of p53 have keratinocytes in two forms: in full length and in an alternatively spliced form lacking exon 5 been identified: p73, and KET. The gene we have cloned and identified as CUSP is a splicing (AltCaR). The AltCaR fails to signal calcium stimulated IP3 production. As keratinocytes variant of KET. CUSP is constitutively and abundantly expressed in keratinocytes and is the major differentiate, the ratio of the full length: alternatively spliced CaR declines. Immunostaining of KET isoform expressed in the skin. keratinocyte cultures using a monoclonal antibody against an extracellular epitope of CaR showed Little is known about the functions of the p53-like proteins. In contrast to p53, p73 is not induced decreased expression of CaR proteins during differentiation. Western analysis using the same by UVR and is almost never found mutated in human tumors. In addition, the CUSP protein, antibody specifically detected several bands with molecular weights between 120 and 200 kDa. which lacks a transactivation domain, fails to activate certain p53 reporter genes and fails to effect The level of the 200 and 160 kDa bands decreased as keratinocytes differentiate, while the level apoptosis. Thus, whatever the functions of the p53-like proteins, they are not identical to those of the 120 kDa band increased in the presence of calcium. When cDNAs of full-length CaR or of p53. The purpose of our study is to examine the effect of UVR on CUSP. If CUSP functions AltCaR were transiently transfected into keratinocytes the full-length CaR greatly enhanced the as a p53-like tumor suppressor in the skin, its regulation by UVR, the major initiator of skin calcium-stimulated involucrin expression, whereas the AltCaR had no effect. To understand the cancer, is likely to be important. role of the CaR in vivo during epidermal differentiation we first cloned and sequenced the mouse Cultured human keratinocytes were exposed to 0–250 mJ per cm2 UV13 radiation using a solar CaR cDNAs then examined the changes in morphology and gene expression in the epidermis of simulator. After 24 h, RNA and protein was analyzed by northern and western blots. Comparable mice in which only AltCaR was expressed (Casr–/–). The mouse CaR shares 95% homology loading of lanes was assured by probing for GAPDH and actin expression, respectively. Higher with human CaR on amino acid level and like the human CaR was expressed as both the full doses of UV radiation selectively repressed CUSP expression, to the extent that CUSP mRNA length and the alternatively spliced forms. Immunolocalization and in situ hybridization of the and protein were virtually undetectable at the 250 mJ per cm2 UV13 dose. In contrast, p21, a epidermis using antibodies and cDNA probes that detect both forms of CaR showed a suprabasal downstream effector which is induced by p53, was found to be increased at the higher UV13 doses. expression of CaR in both Casr–/– mice and its wild-type (Casrϩ/ϩ) littermate. RT-PCR This study shows that the p53-like protein, CUSP, not only is not induced by UVR but is analysis on the CaR mRNA in these animals revealed that the Casr–/– mice expressed only the selectively downregulated by UVR. We propose that CUSP functions in an antagonistic manner AltCaR, while Casrϩ/– mice expressed both forms in both epidermis and kidney. However, to p53, and that the repression of CUSP expression by UVR may allow p53 to function effectively. while the kidney of the Casrϩ/ϩ mice expressed only the full-length CaR, the epidermis Thus, CUSP is likely to play an important role in the cutaneous defense against cancer formation. expressed both forms. Morphological study on the epidermis of the Cars–/– mice showed an expansion of the interface between stratum corneum and stratum granulosum, evidence of abnormal granule morphology and abnormal lamellar body secretion. We next examined the expression of late terminal differentiation marker genes, loricrin and profilaggrin, by immunohistochemistry and in situ hybridization. Both studies showed a reduced expression of loricrin and profilaggrin in the epidermis of Cars–/– mice. These data indicate that the full length CaR is essential for normal epidermal differentiation presumably by mediating the calcium signal required for keratinocyte differentiation. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 559

217 218 Activation of Protein Kinase C by Ultraviolet Radiation Triggers Apoptosis of Human Keratinocyte Epidermal Growth Factor Receptor Phosphorylation Induced by Glutathione-Depleting Agent and Involves Cross-Talk with Caspases n-Ethyl-maleimide is Mediated by H2O2 in Cultured Keratinocytes M. Denning, B. Nickoloff and Y. Wang D. Peus, A. Meves, A. Beyerle and M. Pittelkow Department of Pathology, Loyola University Medical Center, Maywood, Illinois Department of Dermatology and Biochemistry & Molecular Biology, Mayo Clinic, Rochester, Min- Induction of apoptosis by ultraviolet (UV) radiation is an important mechanism for the elimination neapolis of sun-damaged keratinocytes at risk of becoming transformed. The UV radiation death effector H2O2 has been shown to induce growth factor receptor phosphorylation in different cell types. pathway involves activation of the caspase protease cascade, involving sequential activation of We have recently identified H2O2 as an important mediator of UVB-induced epidermal growth initiator caspases (i.e. caspase-8) and effector caspases (i.e. caspase-3). UV radiation also stimulates factor receptor (EGFR) phosphorylation in human keratinocytes. To further characterize the role δ δ the activation of protein kinase C01 (PKC01 ) by caspase dependent proteolysis, in which of H2O2 as a cell-generated mediator, we have examined EGFR phosphorylation induced by n- cleavage of PKC01δ by caspase-3 generates a constitutively active PKC01δ catalytic domain. ethylmaleimide (NEM), a thiol alkylating agent. NEM rapidly depleted keratinocytes of glutathione, We examined the role of PKC activation in the UV radiation death effector pathway of normal the most abundant intracellular thiol that protects cells against oxidative stress. Intracellular H2O2 human keratinocytes and the immortalized HaCaT cell line. Inhibition of PKC activity with the production rapidly increased in response to NEM exposure in a time-dependent manner as PKC inhibitor GF109203X protected normal keratinocytes (76% inhibition) and HaCaT cells analyzed by laser scanning confocal microscopy and flow cytometry. NEM also demonstrated (86% inhibition) from UV radiation-induced apoptosis. Inhibition of PKC also partially blocked time-dependent stimulation of EGFR phosphorylation which was initially observed within 5–15 the cleavage of PKC01δ and caspase-3, and reduced the activity of caspase-3 in both cell types. min. The antioxidants n-propyl-gallate and ascorbic acid-6-palmitate strongly inhibited intracellular δ In addition, expression of the catalytic domain of human PKC01 induced apoptosis of normal H2O2 generation as well as EGFR phosphorylation in a concentration-dependent manner. human keratinocytes and HaCaT cells, as assessed morphologically by bleb formation, chromatin Overexpression of catalase by electroporation of normal keratinocytes also protected against NEM- condensation, and nuclear fragmentation. Transfection of the PKC01δ catalytic domain into induced EGFR phosphorylation. These findings demonstrate that NEM-induced glutathione HaCaT cells also triggered ultrastructural hallmarks of apoptosis. Thus, PKC plays a dual role in depletion is one of the sentinel upstream mechanisms regulating EGFR phosphorylation and the UV-induced death effector pathway, being involved in stimulating caspase activation, and itself cellular levels of H2O2. The results further establish H2O2 as an important second messenger that being activated by caspase proteolysis. These results indicate that PKC activation is required for is likely to be involved in the regulation of other cellular phosphorylation and signaling events UV radiation-induced apoptosis of human keratinocytes, and highlight novel cross-talk between in keratinocytes. the PKC and caspase signaling pathways.

219 220 Nitric Oxide in Stevens Johnson/Toxic Epidermal Necrolysis Cyclosporin A and Calcineurin – Related Pathways in Control of Keratinocyte Growth L. Lerner, A. Qureshi, V. Reddy and E. Lerner and Differentiation Departments of Pathology and Dermatology and the Cutaneous Biology Research Center, T. Seki, L. Bolgan and G. P. Dotto Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts Cutaneous Biology Research Center, MGH/Harvard Medical School, Charlestown, Massachusetts Stevens Johnson Syndrome (SJS) and toxic epidermal necrolysis (TEN) are severe drug reactions Calcineurin is a calcium/calmodulin dependent serine phosphatase with a well characterized characterized by apoptosis and necrosis. Nitric oxide (NO) can cause apoptosis and necrosis. The function in cells of the immune, nervous and muscular systems. Cyclosporin A (CsA) and FK506 inducible form of nitric oxide synthase (iNOS) can generate large amounts of NO and has been are two specific inhibitors of calcineurin activity, which exert an interesting side-effect in the skin, described in human skin. It is proposed that a large burst of NO causes the epidermal necrosis in induction of hypertrychosis, which is unrelated to their effects on the immune system. We report SJS and TEN. here that treatment of keratinocytes with CsA under well defined culture conditions inhibits the To test this hypothesis, skin biopsies obtained from five patients with SJS/TEN were examined expression of a specific set of suprabasal differentiation markers as well as of p21WAF1/Cip1, a for the presence of iNOS. RT-PCR and immunoperoxidase staining were used to detect iNOS cyclin kinase inhibitor associated with keratinocyte differentiation. In parallel with inhibition of mRNA and protein, respectively. mRNA for iNOS was found in all five patients by RT-PCR. the endogenous gene, activity of the p21 promoter in calcium-induced keratinocyte differentiation Immunoperoxidase staining showed strong expression of iNOS in the epidermis and in inflammatory is suppressed by CsA as well as FK506. p21 promoter activity is also suppressed by direct expression cells in the upper dermis in all cases. This expression was most prominent in the lower epidermis of a dominant negative calcineurin mutant, while it is significantly induced by expression of a in four cases, corresponding to the areas of most severe necrosis observed on the diagnostic constitutively active form of calcineurin or the regulatory B subunit alone. Besides p21, increased biopsies from these patients. In the fifth case, full thickness epidermal necrosis was found on the calcineurin expression upregulates the promoter activity for a late keratinocyte differentiation diagnostic biopsy and diffuse epidermal staining for iNOS was present. marker, loricrin, while it has no effect on the promoter for an earlier stage marker, involucrin. These data demonstrate that high levels of iNOS are expressed in the skin of patients with SJS Growth of keratinocytes is partially suppressed by calcineurin expression, and this occurs only in and TEN. The presence of iNOS is consistent with the hypothesis that NO mediates epidermal wild type but not p21-deficient cells. Thus, calcineurin is likely to play an important role in necrosis and, perhaps in association with Fas and Fas ligand, epidermal apoptosis. These results transduction of the signal which leads to keratinocyte growth arrest and differentiation. suggest that iNOS is a potential target for therapeutic intervention in the treatment of SJS and TEN.

221 222 Differential Expression and Function of Cell Cycle Regulatory Proteins During Keratinocyte Skn-1a POU Transcription Factor Enhances Terminal Differentiation in HACaT Cells (KC) Proliferation, Growth Arrest, Differentiation, Replicative Senescence (RS), and Apoptosis J. Hildesheim, R. Foster, U. Kuhn and J. Vogel V. Chaturvedi, J. Qin, M. Denning, D. Choubey, P. Bacon and B. Nickoloff Dermatology Branch, NCI, NIH, Bethesda, Maryland Department. of Pathology, Loyola University, Chicago, Illinois POU transcription factors (TF) regulate cell differentiation and are often expressed in a cell- Once a proliferating KC migrates upward from the basal cell layer, it becomes growth arrested specific manner. However, a biological role for the epidermal-specific POU TF, Skn-1a, in (GA) and begins a differentiation pathway involving keratin-1 (K1) expression, that may culminate keratinocyte (KC) differentiation has not been defined. We and others have previously demonstrated in terminal differentiation, senescence or apoptosis. While orderly stratification maintains constant that Skn-1a can transactivate promoters of KC differentiation marker genes, such as keratin (K) epidermal thickness, molecular events regulating this process are poorly understood. Therefore, 10, small proline-rich protein 2A (SPRR-2A), HPV-1a and HPV-18 long control regions. We we analyzed expression of proteins that control the cell-cycle in both normal KC, as well as have also demonstrated that the critical transactivation domain of Skn-1a resides in the C-terminal immortalized KC (i.e HaCaT). Semi-confluent proliferating KC express low nuclear levels of the region. To determine if Skn-1a can regulate KC differentiation, stratification assays were performed cyclin-dependent kinase inhibitors (CDKI) p15, p16, p21, p27. During confluence-induced GA with HaCaT human KC cell lines. HaCaT do not normally express Skn-1a and do not stratify there were elevated levels of all these proteins; whereas in KC that undergo RS (passages 4–5), in vitro. HaCaT transduced with Skn-1a formed a thickened epithelium in raft cultures with p16 is upregulated. Reversible GA induced by TGF-01β in KC leads to elevated p15, whereas increased numbers of dyskeratotic cells containing pyknotic nuclei than control HaCaT transduced irreversible GA induced by elevated Ca2ϩ, IFN-01γ, phorbol ester, or IFN-01γ plus TPA induced with 01β-galactosidase. Additionally, Skn-1a-expressing HaCaT demonstrated increased expression predominantly p21 and p27. In contrast to KC, HaCaT cells have a very different profile of of the terminal differentiation markers KC transglutaminase and SPRR-2A, consistent with a CDKIs. HaCaT cells had no detectable p16, and after Ca2ϩ exposure, IFN-01γ, TPA, IFN-01γ more differentiated phenotype. PCNA staining of raft cultures at different time points suggested plus TPA exposure, or upon confluence, no p16 was induced with only minimal increase in p21 that the increased cellularity correlated with increased HaCaT proliferation. The dyskeratotic cells and p15. While both proliferating KC and HaCaT cells were susceptible to UVB induced apoptosis had positive staining in the TUNEL assay, suggesting that they were undergoing apoptosis. (30mj, 24 h), KC that underwent irreversible GA or RS were resistant to induction of apoptosis. Expression of an extensive panel of genes that regulate apoptosis was assessed by RNase protection. The antiapoptotic response of the GA KC or senescent KC did not correlate with K1 expression. Interestingly, two antiapoptotic genes, TRAF-2 and TRAF-4, were consistently down regulated Even though HaCaT cells had a comparable antiproliferative response to high Ca2ϩ or IFN-01γ in Skn-1a-expressing HaCaT, compatible with increased programmed cell death. These results plus TPA, these pretreatments did not produce an antiapoptotic phenotype.WhenHaCaT cells suggest that Skn-1a promotes KC terminal differentiation by up-regulating genes associated with were transfected with p16 or p21containing plasmids, they became resistant to UVB induced cornification, and may also decrease expression of ‘‘survival’’ genes in epidermal cells. apoptosis. These results indicate that while immortalized HaCaT cells can be growth arrested, they remain highly susceptible to UVB induced apoptosis. Both reversible and irreversible GA arrested KC preferentially express p21, whereas senescent KC preferentially express p16. Transfec- tion of HaCaT cells with either p16 or p21 produced an apoptotic resistant phenotype, and under no conditions was the antiapoptotic phenotype dependent on early differentiation of KC. These results clearly indicate that differentiation is not required to block apoptosis, and that p16 and p21 participate in discrete pathways differentially regulating the response of KC undergoing growth arrest, senescence and apoptosis. 560 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

223 224 Mouse Periplakin: Genomic Cloning and Gene Targeting Skin Re-Epithelialization from the Hair Follicle in Mouse Keratin 6a Deficient Mice Y. Ryoo, K. Li, S. Aho, B. Cho, J. Klement and J. Uitto S. Wojcik, D. Bundman and D. Roop Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Philadelphia, Departments of Cell Biology and Dermatology, Baylor College of Medicine, Houston, Texas Pennsylvania; Department of Dermatology, School of Medicine, Keimyung University, Taegu, Mouse keratin 6 (MK6) has two highly homologous isoforms (MK6a and MK6b) which are both Korea induced in interfollicular epidermis in response to stress, most notably in wound healing. In Plakin family of proteins, which serve an important function within the cytoskeletal-associated normal hirsute skin MK6a and MK6b are constitutively expressed only in the innermost single attachment structures, also serve as autoantigens in malignancy-associated blistering disease, cell layer of the outer root sheath (ORS) of hair follicles. To determine the relative contribution paraneoplastic pemphigus. In this study, we have cloned mouse periplakin (mPPL), a 204-kDa of the two MK6 isoforms to the ability of keratinocytes to re-epithelialize a wounded area, we member of the plakin family of proteins. The deduced polypeptide contained 1752 amino acids generated mice deficient for MK6a using homologous recombination in embryonic stem (ES) including amino-terminal globular domain, a central coiled-coil rod domain and carboxyl-terminal, cells. The ES cells were targeted using a replacement vector that removed the entire MK6a coding encoded by a total of 22 exons. Comparison of mouse and human periplakin sequences region from the genome. MK6a knockout mice did not show MK6 induction in the basal layer demonstrated high (84.8%) homology. A mPPL targeting vector was constructed from mPPL01λ of the epidermis or the outer ORS of the hair follicles, demonstrating that the MK6a isoform is genomic DNA. An 8-kb XhoI – XbaI fragment, spanning the exons 17–21, and some parts of responsible for inducible MK6 expression in the basal layer of the epidermis and the outer ORS, exon 22nd, was replaced with a 1.8-kb fragment containing a neomycin resistance gene under whereas induction of MK6b is restricted to the suprabasal layers of the epidermis. Based on this the control of pgk1 promoter. The newborn pups are clinically normal and detailed evaluation of differential expression pattern we decided to analyze re-epithelialization of wounded skin from the epidermis is in progress. The development of periplakin deficient mice aids to elucidate the keratinocytes residing in the ORS in MK6a deficient mice, using wild-type littermates as controls. role of periplakin in epithelial tissues. The epidermis was wounded by tape-stripping the shaved, depilated skin, which removes the suprabasal keratinocytes and kills the basal layer. The dead basal cells do however, remain attached to the basement membrane and a fibrin clot then forms underneath the old basal layer. Re- epithelialization at the center of the wound can only occur from of the hair follicles. The keratinocytes of the ORS proliferate and migrate out of the hair follicle, forming a new epidermis under the scab. Preliminary analysis of this process in MK6a deficient mice indicates that, while these mice eventually heal, they do exhibit a delay in re-epithelialization from hair follicles.

225 226 cDNA Microarray Screening for Genes Differentially Expressed in Keratinocyte Stem Cell Semaphorin III Signaling in Skin Enriched Populations M. Yaar, K. E. Traber, H. A. Raynham, D. LoPiccolo, M. Patacchiola and B. Gilchrest R. Freiberg, C. Barry, M. Fang, E. Alvarez-Saavedra, P. Robbins, P. Brown and P. Khavari Boston University School of Medicine, Boston, Massachusetts VA Palo Alto and Stanford University, Stanford, California Semaphorin III (sema III) is a member of a family of highly conserved cell surface and secreted While epidermal stem cells are a focus of great interest, the characterization of these cells has thus proteins that transmit a chemorepulsive signal to developing neurites. Because melanocytes (MC) far proven elusive due to a lack of specific keratinocyte stem cell markers. To address this issue, express signalling molecules that are also expressed by neurons, and kertinocytes (KC) express a we used cDNA microarray screening to identify genes specifically expressed in keratinocyte stem variety of neurotrophic factors, we asked whether sema III and its receptor neurophilin play a cell-enriched versus stem cell-depleted cell populations. First, we isolated a stem cell-enriched role in MC–KC interactions in skin. Using RT-PCR followed by sequence analysis of identified population from normal adult human abdominal skin keratinocytes using rapid adhesion to Type transcripts and primers complementary to human-sema III and neurophilin cDNAs, KC were IV human collagen, which yielded approximately 10% of the input cells consistent with prior found to constitutively express sema III mRNA and MC neurophilin mRNA. Western blot stem cell studies. The the stem cell containing population exhibits two cardinal features of stem analysis of total keratinocyte proteins reacted with anti sema III antibodies identified the ~120 cells, namely pluripotency [in this case the ability to regenerate full thickness epidermis] and the kDa semaphorin protein. Immunohistochemical analysis of normal human skin displayed epidermal capacity for sustained self-renewal. mRNA was isolated from the stem cell-enriched population staining that was particularly strong in the differentiated KC of the upper epidermal layers. The and compared to the stem cell-depleted population from the same patients by cDNA microarray staining disappeared when the antibodies were preabsorbed with the peptide used for their hybridization using approximately 17,500 arrayed genes. Dramatic differences in global patterns generation. Because these results suggest that sema III is more prevalent in differentiating KC we of gene expression were apparent, with more than 1200 genes exhibiting Ͼ200% changes in gene examined the effect of 12-o-tetradecanoylphorbol-13-actate (TPA) (50 ng per ml) and CaCl2 (1.8 expression levels between stem cell enriched and depleted populations. A total of 51 genes which mM), two known mediators of KC differentation, on sema III levels in cultured KC. Both signals were either strongly expressed [33 genes] or entirely absent [18 genes] in the stem cell-enriched increased sema III protein in a time dependent manner with a more pronounced effect observed population were selected for further study after confirmation by northern blot analysis. Strongly in TPA treated cultures. To determine if sema III affects MC dendrite extention, anti sema III expressed genes include those implicated in preventing apoptosis, in control of the cell cycle and antibodies or control IgG were added to KC/MC cocultures. In cultures treated with anti sema transcription and in mediating signal transduction and cell adhesion, however, the majority of III antibodies, MC displayed a more extensive dendritic network as compared to cultures treated genes identified are previously uncharacterized ESTs representing genes of unknown function. with control antibodies. Our results demonstrate that sema III is present in differentiated KC both These data suggest that significant global differences in gene expression exist between stem and in vivo and in vitro, and that MC express the receptor for sema III. This establishes another parallel nonstem cells in the epidermis and identify candidate genes for potential use as keratinocyte stem between MC and neuronal signaling pathways and suggests that the preferential melanosomal cell markers and as probes for further study of stem cell biology. transfer to KC of the lower epidermal layers may be due in part to the chemorepellent effect of sema III dendrite extension to supra-basilar layers.

227 228 The Endogenous Angiogenesis Inhibitor Thrombospondin-2 is Expressed in Basal Keratinocytes Effective Use of Photoimmunotherapy in the Treatment of Severe Acute and Chronic Graft- of Normal Epidermis In Vivo and in Epidermal Keratinocytes In Vitro Versus-Host Disease L. Riccardi, M. Streit, P. Velasco, L. Brown,* J. Lawler* and M. Detmar R. Knobler and the Vienna ECP-Team Cutaneous Biology Research Center, Department of Dermatology, Massachusetts General Hospital Bone Marrow Transplantation Unit, Division of Immunology, Allergy and Infections Diseases, and Harvard Medical School, Boston, Massachusetts; *Department of Pathology, Beth Israel Division of Special and Environmental Dermatology, Department of Dermatology, AKH, Deaconess Medical Center, Boston, Massachusetts Vienna, Austria Thrombospondin-2 (TSP-2) is a new member of the thrombospondin family of matricellular Extracorporeal Photoimmunotherapy (Photopheresis, ECP) has been shown to be effective in the proteins. Recently, it was shown that TSP-2 deficient mice were characterized by increased treatment of a number of T-cell-mediated diseases such as cutaneous T-cell lymphoma (CTCL) vascular density in the skin. The aim of our study was to investigate whether TSP-2 is expressed and rejection of organ (heart, kidney, lung) transplantation. Twenty-one patients (10 men and 11 in normal skin, using ribonuclease protection assays, and to localize TSP-2 expression to distinct women) are presented who after presentation with hematological malignancies (median age of cellular compartments, using in situ hybridization and immunohistochemistry. For immunohisto- presentation: 36 y, range 25–55 y) had received bone marrow grafts from sibling (n ϭ 12) or chemical analysis, we developed a polyclonal rabbit antihuman TSP-2 antibody, directed against unrelated (n ϭ 9) donors. Six patients had acute graft-versus-host disease (GvHD) grade II to III a N-terminal sequence of human TSP-2 that shows the least homology with the related molecule not responding to cyclosporine A (CSA) and prednisolone when referred to photoimmunotherapy. TSP-1. The specificity of this antibody was confirmed by Western blot analyses. Ribonuclease In 15 patients, 2–24 mo after bone marrow transplantation (BMT), extensive chronic GvHD with protection assays of total cellular RNA detected marked TSP-2 mRNA expression in extracts of involvement of skin (n ϭ 15), liver (n ϭ 10), oral mucosa (n ϭ 11), ocular glands (n ϭ 6), and normal mouse skin. In normal human adult skin as well as in neonatal foreskins, prominent thrombocytopenia (n ϭ 3) developed and was unresponsive to conventional therapy, including immunohistochemical staining was observed in the basal layer of the epidermis, whereas no steroids. All patients were treated with ECP on two consecutive days every 2 wk for the first 3 staining was detected in suprabasal keratinocytes. In the dermis, weak staining of blood vessels mo and thereafter every 4 wk until resolution of GvHD. ECP was tolerated well without any and some dermal cells was observed. In situ hybridization with specific antihuman TSP-2 antisense significant side-effects. After a median of 14 cycles of ECP, acute GvHD resolved completely in riboprobes confirmed the immunohistochemical findings: TSP-2 mRNA expression was localized four of six patients (67%) and partially in another two patients. Cutaneous chronic GvHD to the basal epidermal layer, sometimes in a patchy distribution, as well as to some dermal completely resolved in 12 of 15 (80%) patients. Contractures of knees and elbows due to fibroblasts and blood vessels. In vitro studies confirmed TSP-2 expression in both human and scleroderma resolved partially. Oral mucosal ulcerations resolved in all patients. Seven of 10 mouse keratinocytes, using northern and western blot analyses. These findings identify TSP-2 as patients (70%) with liver involvement had complete response after ECP. After discontinuation of a new marker for basal epidermal keratinocytes. While the function of epidermal TSP-2 remains ECP, no severe infections were observed. Based on our findings we conclude that ECP can be to be established, it likely contributes to the anti–angiogenic barrier that prevents vascularization used as a safe and effective adjunct therapy for acute as well as extensive chronic GvHD with of normal epidermis. cutaneous and visceral manifestations, refractory to standard immunosuppressive treatment. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 561

229 230 Induction of Apoptosis in Skin-Infiltrating Mast Cells by High-Dose Ultraviolet A-1 (UVA-1) UV Activates NF-01κB Through an Antioxidant-Sensitive Pathway that is Independent of Radiation Phototherapy in Patients with Urticaria Pigmentosa Cytokines in Human Keratinocytes and Skin In Vivo H. Stege, M. Budde, V. Ku¨rten, T. Ruzicka and J. Krutmann Z. Wang, Y. Wan, J. Voorhees and G. Fisher Department of Dermatology, University of Du¨sseldorf, Germany Department of Dermatology, University of Michigan, Ann Arbor, MI. In previous years, the mainstay in the treatment of patients with urticaria pigmentosa (UP) has Transcription factor NF-01κB regulates expression of numerous proinflammatory genes. Activation been photochemotherapy with oral administration of 8-methoxypsoralen and subsequent ultraviolet of NF-01κB by cytokines requires degradation of its cytoplasmic inhibitor I01κB. Phosphorylation A radiation exposure (PUVA). PUVA therapy is effective, but does not decrease the number of of I01κBbyI01κB kinase (IKK), targets I01κB for proteasome-mediated degradation. We skin-infiltrating mast cells and cessation of PUVA therapy is therefore associated with a relapse of investigated mechanisms by which UV irradiation activates NF-01κB in human skin in vivo and symptoms after only 6–8 mo in almost all patients. More recently, high-dose UVA-1 phototherapy cultured human keratinocytes (KC). In human skin, UV irradiation caused rapid translocation of (340–400 nm; 130 J per cm2 UVA-1 per exposure) was found to be effective for the treatment NF-01κB (p65) from the cytoplasm, where it is sequestered bound to I01κB, to the nucleus, in of UP (Lancet 347:64, 1996). In continuation of these studies we now report that UVA-1 KC throughout the epidermis. Surprisingly, this occurred in the absence of any reduction in the phototherapy differs from PUVA by inducing significantly longer remission periods. High-dose level or cytoplasmic localization of I01κB. Although we found that human skin expressed both UVA-1 phototherapy induced a prompt (after three exposures) and complete (after 10 exposures) IKK-01α and IKK-01β, UV irradiation did not stimulate IKK activity, consistent with failure of improvement of skin as well as systemic symptoms in 20 out of 20 patients with histologically UV to degrade I01κB. Similarly in KC, UV irradiation did not stimulate IKK activity or I01κB proven UP. Follow-up studies revealed remission periods of 12 mo in 92.8%, of 15 mo in 69.2%, degradation, although UV did cause rapid nuclear translocation of NF-01κB (p65). The actions and of 24 mo in 55.5% of these patients. Immunohistochemical analysis of biopsies obtained prior, of UV are largely mediated by reactive oxygen species. Therefore, we determined the effects of during and after UVA-1 phototherapy demonstrated that clinical improvement was associated with the antioxidant N-acetyl cysteine (NAC) on UV versus IL1-induced NF-01κB activation. a significant decrease in the number of skin-infiltrating mast cells in these patients. Accordingly, Pretreatment of KC with NAC completely blocked UV-induced NF-01κB translocation. In after only three exposures, mast cell numbers were decreased by up to 50%. Mast cell depletion contrast, NAC pretreatment did not prevent IL-1 induced I01κB degradation or NF-01κB was preceded by the induction of apoptosis in these cells, as was demonstrated by employing a translocation. These data demonstrate that UV and cytokines activate NF-01κB through distinct double-staining technique allowing the simultaneous detection of apoptotic (TUNEL method) as pathways. Further elucidation of the antioxidant-sensitive pathway by which UV activates NF- well as tryptase-positive (01α-mast-cell-tryptase Mab; immunofluorescence) cells. These studies 01κB will provide novel targets for mitigating UV damage to human skin. indicate that UVA-1 phototherapy induced apoptosis in skin-infiltrating mast cells and subsequent mast cell depletion are responsible for the long-lasting clinical improvement that is induced in UP patients by high-dose UVA-1 phototherapy. They also suggest that high-dose UVA-1 phototherapy is the treatment of choice for UP.

231 232 Identiﬁcation of a Novel Signal Transduction Pathway Involved in Ultraviolet A Radiation UV Light Effects on Apoptosis Regulatory Proteins, Bax and Bcl-2, in Melanoma (UVAR)-Induced Gene Expression D. Marr, Y. Shellman, J. Gendall, M. Fujita and . DA Norris S. Grether-Beck, G. Bonnizi,* H. Schmitt, H. Sies,† J. Piette* and J. Krutmann Department of Dermatology, University of Colorado Health Sciences Center, Denver, Colorado Clinincal & Experimental Photodermatology, Department of Dermatology, †Institute of Molecular Apoptosis (programmed cell death) is a physiologic mechanism by which the human body Biochemistry, University of Du¨sseldorf, Germany; *Laboratory of Molecular Chemistry, University maintains homeostasis. Apoptosis often serves as a response to stress-induced cell damage in order of Liege, Belgium to prevent outgrowth of aberrant cell populations. Antipodal modulation of the apoptosis regulatory We have recently shown that UVAR-induced keratinocyte (KC) gene expression is mediated proteins Bax and Bcl-2 by ultraviolet radiation (UVR) has been described for numerous cell types. through the formation of ceramides from cell membrane sphingomyelin as an initial step. In the The purpose of this study was to determine the effects of UVR on the expression of these present study the mechanism by which UVAR induces ceramide generation was assessed. After apoptosis regulatory proteins in radial growth phase malignant melanoma (WM35), cells which metabolic labeling of cells with [3H]palmitic acid, lipid extracts from irradiated normal human are continuously exposed to UVR in vivo. As a negative control, dominant negative p53 transfected KC were analyzed for the formation of ceramides by thin layer chromatography (TLC). Ceramides WM35 melanoma cells (WM35dnp53) were used since the UV effects on Bax and Bcl-2 are p53 were generated in KC upon UVAR exposure in a dose-and time-dependent manner. UVAR- dependent. Mutant ras WM 35 transfectants (WM35mnras) were also studied because activated 1 induced ceramide formation was mediated through singlet oxygen ( O2) generation, because it ras maintains high levels of bcl-2 expression. Melanoma cell lines were grown in monolayer 1 was completely prevented, when KC were irradiated in the presence of O2 quenchers (sodium culture, subjected to varying doses of UVR from a solar simulator, and maintained in culture for 1 azide, vitamin E), and because it could be mimicked by treating unirradiated cells with a O2- 24 additional hours. The cells were then harvested and protein expression was measured using α 2 generating system (thermal decomposition of NDPO2). The cytokines TNF01 and IL-1 are western blotting. Treatment with UVR (up to 1000 mJ per cm ) resulted in decreased Bcl-2 known to induce ceramide-mediated gene expression through activation of sphingomyelinases expression even in the WM35mnras cells, but less decrease was seen in the dominant negative (SMase) that cause hydrolysis of sphingomyelin and thereby generate ceramides. Accordingly, p53 (WM35dnp53). This effect has been previously described in many types of cells. However, TNF01α or IL-101α induced (4-fold) acidic and neutral SMase activity in KC, as was assessed in expression of Bax was also decreased upon UVR treatment in both the control and Ras-transfected KC protein extracts that were incubated with C14-sphingomyelin. In contrast, SMase activity melanoma cell lines, but not in the WM35dnp53 line. This is in contrast to the UVR induced remained unaltered when KC were treated with UVAR or NDPO2, although both stimuli increases in Bax expression in other cell types. Thus, the pattern of regulation of Bax and Bcl-2 induced (3-fold) ceramide formation within the same experiment. In order to test whether UVAR/ in melanoma cells is different than described in other cell lines, and UV induces down regulation 1 O2 generates ceramides independent of SMase activity, sphingomyelin-containing liposomes were of both pro and antiapoptotic proteins. This may be one of the reasons for the relative resistance irradiated. In this SMase-free system, UVAR as well as NDPO2 treatment caused the formation of melanoma cells and melanocytes to UVR induced apoptosis compared to other cell lines, and of ceramides, as was measured by TLC and conﬁrmed by mass spectroscopy. Our studies indicate may offer some survival advantage to these cells in the human epidermis. 1 the existence of a previously unrecognized, nonenzymatic, O2-mediated pathway, by which UVAR initiates the signal transduction cascade leading to increased KC gene expression.

233 234 The Xeroderma Pigmentosum Group C Gene Leads to Preferential Repair of UV-Induced Activation of the Tyrosine Kinases and p53 by Thymidine Dinucleotide: Potential Upstream Cyclobutane Pyrimidine Dimers Regulation by the ATM Kinase S. Emmert, N. Kobayashi, S. G. Khan, and K. H. Kraemer M. Eller, M. Barratt* and B. A. Gilchrest National Cancer Institute, Bethesda, Maryland Boston University School of Medicine, Boston, Massachusetts; and *Unilever Research, Edgewater, We investigated the contribution of the xeroderma pigmentosum group C (XPC) gene to DNA New Jersey repair following UV exposure in human cells. We stably transfected DNA repair deficient XPC DNA damage is known to induce responses in mammalian cells such as altered gene expression, cells (XP4PA-SV-EB) with normal XPC cDNA and selected two clones (SE1 and SE2). DNA repair, cell cycle inhibition and programmed cell death, but the signal transduction pathways Transfection of pRSVcat exposed to 1000 J per m2 UVC resulted in 3% reporter gene expression involved are poorly understood. One protein thought to sense and/or respond to DNA strand in XP4PA cells, 10% in SE1, 23% in SE2, and 45% in repair-proficient cells compared to untreated breaks is ATM (for ‘‘mutated in the disease ataxia telangiectasia’’). Although the exact function of control plasmids. Post-UVC cell survival was low for XP4PA cells, intermediate for SE1 cells, ATM is unknown, it shares homology with a family of kinases and has been shown to phosphorylate and normal for SE2 cells. The XP4PA cells had undetectable XPC-mRNA by northern blot p53 in vitro and to activate the c-Abl kinase in vivo in response to ionizing radiation. We have analysis while SE1 cells had nearly normal and SE2 cells had elevated levels compared to normal previously reported that many of the effects of DNA damage can be mimicked in vitro and in vivo cells. Using an enzyme linked immunosorbent assay and damage specific monoclonal antibodies by small DNA fragments, particularly thymidine dinucleotide (pTpT). These effects include (Photochem Photobiol, 1991; 54:225) we assessed the repair kinetics of cyclobutane pyrimidine increased melanogenesis, activation of p53, enhanced DNA repair, induction of TNFa synthesis, dimers (CPD) and (6–4) photoproducts (6–4PP). By 24 h the XP4PA cells could not remove and inhibition of contact hypersensitivity. In order to elucidate more precisely the mechanism of CPD or 6–4PP from the global genome. SE1 cells were also completely unable to repair 6–4PP action for pTpT, normal human keratinocytes were treated with either pTpT or diluent alone for but their 25% removal of CPD at 24 h after 30 J per m2 UVC was nearly normal. SE2, like three hours, then cell lysate was collected and incubated with an array of membrane-attached normal cells, repaired 30% of CPD and 85%–90% of 6–4PP by 24 h. We performed similar peptide substrates for selected kinases in the presence of [g-32P] ATP. Substrate phosphorylation experiments with partially corrected XPD cell lines (Cancer Res 1994; 54:3837) but detected no was detected by autoradiography and scintillation counting. Of the 17 kinases assayed, three were selectivity in DNA photoproduct repair in the XPD cell lines. XPC is involved in early damage affected by pTpT treatment. The activity of ERK/MAP kinase, commonly up-regulated by recognition during the nucleotide excision repair process along with XPA. Previously, XPA has growth factors was reduced approximately 50% while the c-Abl and c-Src tyrosine kinases activated been shown to preferentially repair 6–4PP (Cancer Res 1995; 55:6152). Here, we demonstrate that by genotoxins such as UV light were stimulated 30% and 40%, respectively. Because ATM has XPC gene expression leads to preferential repair of CPD in the global genome DNA. been implicated as an upstream activator of c-Abl and p53, we examined the effect of pTpT in AT cells. Fibroblasts from AT patients and normal controls were treated with 100 mM pTpT or diluent and the level of the p21 mRNA, transcriptionally up-regulated by p53, was determined by northern blot hybridization. While pTpT increased the level of p21 mRNA 2–3-fold within 24 h in normal cells, the level of p21 message was unchanged in the AT cells. Together these data suggest that the effects of pTpT are mediated at least in part through the ATM protein, with subsequent activation of specific signaling pathways that include activation of c-Abl and p53. A better understanding of the mechanism(s) by which DNA fragments such as pTpT stimulate photoprotective responses such as melanization and DNA repair in the absence of DNA damage. 562 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

235 236 A New Mechanism for Ultraviolet Light Induced Progression of Melanoma: UVR Induces Photocarcinogenesis in Ornithine Decarboxylase Overexpressing Mice Release of the Protein MIA, A Detachment Factor for Melanoma H. Mukhtar, A. Gilliam, S. Farouk, S. Katiyar, N. Ahmad and T. O’Brien D. A. Norris, D. Marr, M. Fujita, R. Buettner and A. Bosserhoff Department of Dermatology, Case Western Reserve University, Cleveland, Ohio; and The Department of Dermatology, Univ of Colorado HSC, Denver Colorado; and Department of Lankenau Medical Research Center, Philadelphia, Pennsylvania Pathology and Dermatology, University of Regensburg, Regensburg, Germany To examine the role of ornithine decarboxylase (ODC) in photocarcinogenesis, we conducted Melanoma Inhibitory Activity (MIA) is an 11-kDa protein which was originally described as an short-and long-term ultraviolet B (UVB)-radiation exposure experiments in K5/ODC-transgenic inhibitory factor for growth of melanoma cell lines in culture. It is a potent detachment factor for mice. In this line, a bovine KIII (K5) promoter/regulatory region drives expression of the truncated melanoma cells in culture, interfering with binding to laminin, fibronectin and tenascin. The ODC protein in basal keratinocytes of the interfollicular epidermis as well as the outer root sheath presence of MIA in the serum of progressing melanomas supports its role as a melanoma progression of the hair follicle. After a single UVB exposure of 180 mJ per cm2, transgenic mice developed factor promoting melanoma detachment from basement membrane proteins. The purpose of this very small increases in bifold skin thickness and in ODC activity. In SKH-1 hairless mice, the study was to determine the effect of ultraviolet radiation (UVR) on release of MIA from melanoma most common and highly sensitive model for photocarcinogenesis and in littermate nontransgenic cells and to determine MIA release was influenced activating ras mutations or dominant negative mice, increases in skin thickness and ODC activity were substantial. In long-term experiments, p53 mutation. Melanoma cell lines established in culture for two days were irradiated with UVR mice were exposed to 180 mJ per cm2 of UVB radiation thrice a week for 2 wk (tumor initiating from a solar simulator and 24 h later the supernatant was harvested for assay of MIA levels by a dose). By 30 wk, eight of 20 UVB-exposed transgenic mice developed epidermal tumors. Each capture ELISA assay. MIA secretion into media was adjusted to ng per ml per 105 cells. MIA tumor histologically verified was benign papilloma. Under the UVB-exposure protocol employed, production by the radial growth phase melanoma cell line WM35 transfected with a control SKH-1 hairless or littermate nontransgenic mice did not develop tumors even up-to 50 wk. Each plasmid (WM35neo) was compared to the same cell line transfected with mutated activating H ODC transgenic mouse developed more than 20 pigmented cysts that contained keratinocyte ras (WM35mnH-ras) or with dominant negative p53 (WM35dnp53). The control cell line material with increased keratinocyte melanization. Out of eight transgenic mice, which following (WM35neo) showed progressive increase in MIA release following increasing doses of UVR with UVB exposure were given 1% (wt/vol) 01α-difluoromethylornithine in the drinking water, none a 2-fold increased release at 100 mJ per cm2 (p Ͻ 0.003). There was no significant difference developed epidermal tumors. The formation of pigmented cysts in these mice was also substantially between the WM35neo and WM35mn Ras. Conversely, The WM35dnp53 lines showed very lower with less than 10 per mice on test. These data indicate that ODC overexpression leads to low baseline levels of MIA secretion without induction by UVR. We have identified a p53 clonal expansion of UVB initiated cells to form epidermal cysts and tumors. dependent effect of UVR likely to promote the detachment of radial growth phase melanomas from basement membrane proteins, a unique progression mechanism.

237 238 The Nitroxide Tempol Affords Protection Against Ultraviolet Radiation as Assayed Using a Abortive Repair Intermediates Represent a Stronger Transcriptional Inhibitor than Unrepaired Transgenic Mouse Molecular Model of Cutaneous Photoaging DNA Damage D. Brown, S. Kong, B. Kwak, T. Takeuchi, J. Uitto and E. Bernstein M. Berneburg,*† P. Clingen,* S. Araujo,‡ R. Wood,‡ J. Krutmann† and A. Lehmann* Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Philadelphia, *MRC CMU, University of Sussex, Brighton, UK; †Department of Dermatology, University of Pennsylvania Du¨sseldorf, Germany; ‡ICRF, South Mimms, UK The generation of reactive oxygen species is one of several mechanisms by which ultraviolet Nucleotide excision repair (NER) removes DNA damage in a multistep process. Incision on the radiation damages skin and its components. Tempol, a superoxide dismutase analogue which 3` and 5` sides of the damage is followed by removal, resynthesis and ligation of the repaired readily penetrates cell membranes when administered exogenously, has been shown to provide patch. It is known that cells from the NER deficiency syndromes xeroderma pigmentosum protection against some forms of oxygen-dependent damage. To determine if tempol is able to complementation group D (XP-D) and trichothiodystrophy (TTD) exhibit different transcriptional protect against ultraviolet B-and ultraviolet A-induced damage, we utilized a previously described levels of UV-responsive genes after UV-irradiation. In order to investigate the underlying transgenic mouse model of cutaneous photoaging which contained the entire human elastin mechanism, we carried out in vivo and in vitro studies to assess the rate of incision and complete promoter linked to a chloramphenicol acetyltransferase reporter gene. Skin fibroblasts were removal of DNA damage and correlated this with transcription of the UV-responsive gene ICAM- cultured from the transgenic mice, incubated with 100 mM tempol for 12.5 min followed by 1 as well as survival after UV irradiation in different NER deficient cell lines. Incision and removal exposure to either ultraviolet B or ultraviolet A radiation. The ultraviolet A response was enhanced of DNA damage in XP-D cells (deficient), most TTD (proficient) and cells from patients with by the addition of 1 µg per ml of 8-methoxypsoralen. The cultures were incubated for 24 h at Cockayne Syndrome (proficient) correlated well with transcription efficiency of ICAM-1 and 37°C; the reporter activity was determined by the production of acetylated chloramphenicol from survival, indicating that removal of DNA damage is necessary to allow transcription of UV cell extracts incubated with 14C-chloramphenicol and acetyl-CoA. Tempol provided 41, 74, 69, responsive genes. Cells from a patient with a combined phenotype of XP and CS exhibited the and 42% protection against 0.7, 1.4, 2.7, and 5.5 mJ per cm2 of ultraviolet B and 40%, 86%, and lowest post-UV ICAM-1 transcription and cell survival. Conversely, this cell line showed severely 91% protection against 0.5, 0.75, and 1.0 J per cm2 of ultraviolet A radiation. These results impaired removal of damage. However, the rate of incision was normal, indicating that in this demonstrate the ability of tempol to protect against both ultraviolet B-and ultraviolet A-induced cell line incision around DNA damage is uncoupled from the actual removal of DNA damage. elastin promoter activation. This protection occurs due to the ability of tempol to behave not Taken together, these results indicate that (i) the removal of CPDs critically determines a cells only as a sunscreen, but also a free radical scavenger. Although tempol may in part reduce capacity to transcribe genes involved in a UV-response and (ii) incomplete repair, generating ultraviolet B effects through direct absorption, its negligible absorption of ultraviolet A suggests abortive intermediates, represents a stronger inhibitor for post-UV transcription than unrepaired that the ultraviolet A protection is related to its reactive oxygen species scavenging ability. This DNA damage. compound may be a useful agent in prevention of cutaneous photoaging.

239 240 Loss of p16 Confers a Reduced and Error-Prone DNA Repair Upon Melanoma Cells: A Role of C-Terminal Tyrosine Residues in RXR Function Molecular Link Between Cell Cycle Disturbance, Repair of UV-Induced DNA Damage, and M. P. Santini, P. LaCelle, B. Graff and R. Polakowska Melanoma Risk? Department of Dermatology, University of Rochester, Rochester, New York, New York T. M. Ru¨nger,*† M. Leverkus,‡ M. Castellano§ and P. Parsonsϩ The Retinoid X Receptors (RXR) are ligand activated transcription factors which also function Departments of Dermatology, *Boston University, Massachusetts; Universities of †Gottingen and as essential partners for heterodimerization with retinoid, steroid, and thyroid hormone receptors. ‡Wurzburg, Germany; §Istituto Naz. per lo Studio e la Cura dei Tumori, Milano, Italy; The effect of RXR on transcription is determined by its dimerization partner, which controls ϩQueensland Institute of Medical Researcg, Herston, Australia ligand specificity and hence interaction with either coactivators or corepressors, and which The melanoma suppressor gene p16 plays an important role in cell cycle control. Cell cycle determines the specificity of target promoter binding. We tested the hypothesis that, like other regulation is known to have a profound impact on how cells process DNA damage, as, e.g. nuclear receptors, RXR activity can also be modulated by the action of nonligand signal exemplified by the diverse functions of p53 at the Gl-checkpoint. In order to study the impact transduction pathways activated in response to tissue-specific extracellular stimuli. To investigate of a p16 mutation on the capability of melanoma cells to process UV-induced DNA damage, we the mechanisms underlying the cross-talk between nuclear and membrane receptors and to establish measured nucleotide excision repair and base excision repair in three p 16-deleted melanoma lines whether post-translational modification of RXR may be involved, we replaced each of the three (ME14/02, MM229, HT144), in two p16-intact melanoma lines (A2058, MM603), and in conserved tyrosine residues in the dimerization interface of the RXR ligand binding domain primary, neonatal melanocytes, using host cell reactivation assays. Plasmids were exposed in vitro (LBD) with phenylalanine. Assessment of the properties of the wild type and mutated forms of either to UVB which induces pyrimidine dimers, or to singlet oxygen, which induces oxidative human RXR01β, in the presence and absence of cognate ligands and tyrosine-specific phosphatase base modifications typical for UVA, and were then transfected into the host cells. Repair efficiency and kinase inhibitors, revealed that each of the three conserved tyrosines, to different extents, are was determined by transformation ability of replicated plasmids recovered from the host cells, required for dimerization, cooperative DNA-binding, and transactivation. Interestingly, tyrosine where they had to be repaired by the host cells’ repair enzymes before replication. Repair fidelity mutation induced ligand-dependent activation, by the normally silent homodimer, of RXRE. In was determined by the mutation frequency in the recovered plasmids. With UVB-irradiated addition, dimerization of RXR01β was blocked by the addition of phosphotyrosyl peptide plasmids, the repair was significantly less efficient in p16-deleted, as compared to p16 intact comprising the sequence surrounding the conserved tyrosine residues, and by phosphatase melanoma lines (e.g. 2.1-fold with 0.5 J per cm2 and 13.5-fold with 1.0 J cm2) and equally treatment, highlighting the possibility that tyrosine phosphorylation may regulate the interaction efficient primary melanocytes, while the mutation frequency was 1.8–4.1-fold higher. The repair of RXR with other receptors and accessory transcription factors. We conclude that the three of oxidative, UVA-typical DNA damage was similar in both groups of melanoma lines. The tyrosines of the hRXR01β-LBD are critical determinants of receptor function and that they may reduced and more error-prone repair of DNA photoproducts in p16 deleted melanoma cells be targets of tyrosine kinase-based signaling pathways, perhaps constituting a signal convergence suggests that loss of p16-function renders melanocytes/melanoma cells hypermutable to UVB- point in eukaryotic gene regulation. irradiation. This molecular link between cell cycle disturbance and UV-sensitivity underlines the role of UV-exposure as a risk factor for melanoma, especially for familial melanoma with p16 germ line mutations. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 563

241 242 Retinoic Acid Stimulates Ubiquitination and Subsequent Proteasome-Mediated Degradation of Collagen Gene Regulation by Transforming Growth Factor-01β1 via Smads Involves Modulation Nuclear Retinoid Receptors in Human Keratinocytes: A Novel Mechanism for Attenuation of of Smad Expression Retinoid Signal Transduction Y. Mori, S-J. Chen, W-H. Yuan and J. Varga M. Boudjelal, J. Voorhees and G. Fisher Section of Rheumatology, University of Illinois at Chicago College of Medicine, Chicago, Illinois Dermatology, University of Michigan, Ann Arbor, Michigan Transforming Growth Factor-01β (TGF-01β) induces extracellular matrix production in fibroblasts Retinoids exert their biological effects through their nuclear receptors, RAR and RXR, that through serine/threonine kinase receptors. We have previously shown that Smads are downstream function as ligand-activated transcription factors. The predominant retinoid receptors in human signal transducers of TGF-01β. Here, we studied the mechanism of Smad regulation of COL1A2 keratinocytes are RAR01γ and RXR01α. We investigated the effect of all-trans and 9-cis retinoic activity. Smad expression was examined in primary normal human dermal fibroblasts by northern acid (RA) on RAR01γ and RXR01α levels in human keratinocyte HaCaT cells. Unexpectedly, analysis, RT-PCR, western analysis, and immunohistochemistry. The effect of Smads on COL1A2 both RAR01γ and RXR01α protein levels were substantially decreased in HaCaT cells cultured promoter activity was analyzed by transient cotransfection of Smad expression vectors with type in the presence of all-trans or 9-cis RA. RAR01γ and RXR01α were decreased 50% within 4 h, I collagen (COL1A2) promoter-CAT constructs, or heterologous TK minimal promoter constructs. 70% after 8 h, and remained reduced for at least 24 h after addition of retinoids (n ϭ 3). Pulse- The results showed that several Smads were expressed in unstimulated fibroblasts. TGF-01β1 chase studies revealed that retinoids reduced the half-lives of RAR01γ and RXR01α from 8 h caused a delayed (Ͼ90 min) and time-dependent reduction of Smad3 mRNA expression. After in vehicle-treated to 4 h in retinoid treated HaCaT cells. Therefore, retinoids increased the rate 24 h of TGF-01β1 treatment, Smad3 mRNA was undetectable in the fibroblasts. In sharp contrast, of RAR01γ and RXR01α degradation. We found that proteasome, but not calpain, serine, or Smad7 mRNA was rapidly and transiently up-regulated by TGF-01β. Smad4 mRNA was lysosome protease inhibitors prevented retinoid-induced retinoid receptor loss in HaCaT cells. unchanged. Smad3 – but not Smad2 or Smad7 – was a potent transactivator of COL1A2. However, retinoids did not alter total cellular proteasome activity. Ubiquitination targets proteins Transactivation was mediated through a specific TGF-01β-responsive CAGA sequence of the for degradation by proteasomes. We found that both RAR-01γ and RXR-01α were substrates promoter shown to be a Smad3-binding element. Smad7 abrogated Smad3- or TGF-01βr- for ubiquitination in HaCaT cells (n ϭ 3). Notably, retinoid treatment significantly stimulated Tinduced COL1A2 transactivation. Expression of Smad3 and Smad7, positive and negative ubiquitination of both RAR-01γ and RXR-01α by 2–3 fold. We conclude that retinoids reduce intracellular transducers of TGF-01β signals, is markedly altered by TGF-01β in dermal fibroblasts. RAR01γ and RXR01α levels by stimulating their ubiquitination and subsequent degradation by The findings indicate, for the first time, that TGF-01β modulation of COL1A2 expression via proteasomes. Our studies reveal a novel mechanism for attenuation of retinoid signal transduction, Smads involves not only their ligand-induced phosphorylation and nuclear localization, but also and provide new insights into the molecular basis of retinoid resistance. regulation of their gene expression. Rapid up-regulation of the inhibitory Smad7, and delayed down-regulation of the stimulatory Smad3, suggest the existence of potent autocrine negative feedback loops of TGF-01β signaling.

243 244 In Vivo Activation of the UV-Responsive Signal Transduction Cascade in Human Epidermal Ker- Switching of Keratinocyte Phenotype by a Novel Glutamate Signalling Pathway Analogous to atinocytes Synaptic Transmission in the Central Nervous System T. Kaneko, M. Komine, K. Tamaki, A. Gazel, G. Pintucci, I. M. Freedberg and M. Blumenberg S. Maxfield,* J. Maltman,† G. Kennovin,† C. Bowgen,† M. Raxworthy,† E. Wood,* T. Skerry Department of Dermatology The University of Tokyo, Tokyo Japan and the RO Perelman and P. Genever Department of Dermatology NYU Medical School, New York, New York Department of Biology, University of York, York, UK; *School of Biochemistry and Molecular Although epidermis is the main target of environmental UV illumination, the signal transduction Biology, Leeds University, UK; and †Smith and Nephew Group Research Centre, York Science pathway responsive to UV has been studied almost exclusively in nonepidermal cell types. In such Park, UK cells UV light activates a pathway including sequentially acting protein kinases JNKKK, JNKK Maintaining the integrity of the epidermis is an essential biological function that relies on co- and JNK. When activated, JNK enters the nucleus to phosphorylate and thus activate transcription ordinated alterations in keratinocyte phenotype. A range of growth factors and cytokines has factors such as Elk1, Jun and ATF2. Using epidermal keratinocytes and skin explants as the targets, previously been shown to be important in regulating these changes in cell behaviour. Here we we examined the activation of JNK by UV light. We determined that keratinocytes express JNK1 provide for the existence of a novel glutamate-mediated signalling pathway in epidermal and JNK3 isoforms of JNK, whereas dermal fibroblasts express JNK1 and JNK2. Keratinocytes keratinocytes. Glutamate is the principal excitatory amino acid within the central nervous system also express the JNKK1/SEK1 activator of JNK and a set of 10 different JNKKKs. UV potently and mediates its effects through interaction with a number of diverse receptor subtypes. Specific and rapidly activates JNK1 and JNK3 in cultured keratinocytes. Concurrently, the activated JNK transporters regulate signalling by removing and recycling released glutamate. We have demonstrated enters the nucleus. An Elk1-responsive promoter construct transfected into keratinocytes is strongly that rat and human epidermal keratinocytes express NMDA, AMPA and metabotropic type activated by UV light. In vivo in light-protected human epidermis, JNK protein is present both glutamate receptors, EAAC1 and GLT-1 glutamate transporters and the GRIP membrane in the nucleus and in the cytoplasm, but antibodies specific for the activated form of JNK show clustering protein, with a clearly defined basal cell expression pattern. In skin cryosections, only a weak, diffuse cytoplasmic staining. UV illumination of skin explants causes rapid, strong immunocytochemistry revealed specific expression of the NMDA-type ionotropic glutamate and specific activation and nuclear translocation of JNK proteins. This in vivo activation follows receptor and the high affinity transporter, EAAC1, by basal keratinocytes. This patterning of the same kinetics as the activation in cultured keratinocytes. Our results demonstrate the functional expression was altered dramatically in human scar tissue and during re-epithelialisation of full activation of the UV-responsive signal transduction cascade in human keratinocytes and, more thickness wounds in rats; NMDA receptor expression was selectively lost in migrating epidermis, important, illustrate the usefulness of skin explants for the analysis of regulatory molecular events whereas EAAC1 was markedly upregulated and expressed by basal and suprabasal cells. The in human epidermis in vivo. expression of the GLT-1 glutamate transporter was also detected on suprabasal keratinocytes in regions of hyperproliferation at the wound margins, though it appeared to be absent from resting human epidermis. In addition, we have shown that during embryonic epidermal development, NMDA-type receptors are expressed suprabasally at E14 and E16, but restricted to basal cells by E18. In vitro, the addition of the specific NMDA receptor antagonist, MK-801 (100 µM) to cultures of primary human keratinocytes induced stratification and the formation of cellular networks at low calcium concentrations where control cultures formed monolayers. Our data strongly suggest that a functional glutamate signalling pathway exists in the epidermis. The distinct alterations in expression patterns of glutamate signalling molecules during epidermal differentiation, regeneration and development, suggests that glutamate may be critically important in the cell signalling events which result in the phenotypic switching of keratinocytes. These findings will clearly impact on many areas of skin pathophysisology and suggest that epidermal glutamate signalling proteins represent potentially novel therapeutic targets for the treatment of skin disease and the enhancement of wound healing.

245 246 Dissecting EGFR Signalling Pathways in the Skin The Role of fyn Tyrosine Kinase in Keratinocyte Differentiation M. Sibilia, A. Behrens, A. Fleischmann, L. Stingl, J. Carroll,* F. Watt,* J. Schlessinger† and E. J. T. Seykora and P. Stein F. Wagner The Wistar Institue and The University of Pennsylvania Medical Center, Philadelphia Pennsylvania Research Institute of Molecular Pathology (IMP), Dr Bohr-Gasse, Vienna, Austria; *ICRF, The fyn tyrosine kinase plays a specific role in the terminal differentiation of murine keratinocytes Lincoln’s Inn Fields, London, UK; †Department of Pharmacology, NYU Medical Center, New which cannot be complemented by other related nonreceptor tyrosine kinases. Phorbol ester York, New York treatment or increased extracellular calcium levels are known to activate fyn kinase activity in Depending on their genetic background EGFR null mice die at midgestation, birth or can live murine keratinocytes. In fyn knockout mice, the keratinocytes are deficient in their expression of up to postnatal day 20 and predominantly display defects in the brain and epithelial tissues such keratin 1 and filaggrin when subjected to stimuli that induce differentiation. In order to better as skin (M. Sibilia and E.F. Wagner, Science 269:234–238, 1995; M. Sibilia, J. P. Steinbach, L. understand the role of the fyn kinase in murine keratinocyte differentiation, we have used the Stingl, A. Aguzzi and E.F. Wagner, EMBO J 17:719–731, 1998). The epidermis and hair follicles yeast two-hybrid technique to isolate fyn-interacting molecules from a murine keratinocyte are severely affected in the mutants, which fail to develop a hairy coat. In contrast, mice carrying library. We have isolated a previously unstudied molecule that interacts with fyn and contains a hypomorphic EGFR allele such as waved-2 (wa-2) show no overt abnormalities other than a phosphorylation consensus sequences for this kinase; we term this molecule FIa. FIa also contains wavy coat and curly whiskers, indicating that skin and hair follicle development are particularly a sequence consistent with an SH3 binding site. When this molecule is expressed in the presence sensitive to aberrant EGFR signalling. of fyn, it is phosphorylated. This molecule has high homology to an unpublished human gene In order to investigate the role of EGFR in skin and to identify downstream components of the whose function is unknown. In the N-terminal region, FIa exhibits some homology to STAMs EGFR signalling pathway, we have generated transgenic mice that express a activated form of the which are molecules that associate with janus tyrosine kinases and are involved in cytokine human Son of Sevenless (hSOS) gene under the control of the keratin 5 promoter (K5). The K5 signalling. We are determining what are the molecular determinants responsible for the fyn–FIa promoter directs the expression of hSOS to the same cells that normally express the EGFR. Four interaction, and the role of FIa in fyn signal transduction. transgenic founders were obtained and all developed severe skin diseases ranging from epidermal hyperplasias to papillomas depending on the number of integrated transgenes. These skin phenotypes develop within 10 wk after birth and with 100% penetrance. Genetic experiments reveal that overexpression of hSOS can partially rescue the skin defects, restore hair growth and prolong the lifespan of EGFR mutant mice. Interestingly, the presence of a hypomorphic EGFR allele attenuates the development of hSOS dependent skin tumors. These results provide genetic evidence for cooperation between EGFR and SOS not only during skin development but also during tumor formation. Investigations into the mechanisms that are responsible for tumor formation are ongoing and results from these experiments will be presented. 564 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

247 248 Specific and Redundant Functions of Src and Fyn Kinases in Keratinocyte Differentiation Control Blocking of UVB-Induced Cytokine Production in Langerhans Cells by a 20-bp NFkB Decoy Nuc- S. Cabodi,*†‡ E. Calautti,† P. L. Stein‡ and G. P. Dotto† leotide *Department of Biology, Genetics and Medical Chemistry, University of Turin, Italy; †Cutaneous K. Abeyama, P. R. Bergstresser and A. Takashima Biology Research Center, MGH and Harvard Medical School, Charlestown, Massachusetts; Department of Dermatology, UT South-western Medical Center, Dallas, Texas ‡Wistar Institute, Philadelphia, Pennsylvania Dysregulation of Langerhans cell function is one mechanism by which UVB radiation distorts Tyrosine phosphorylation is an early event required for keratinocyte differentiation. We have cutaneous immunity. In this study we tested the hypotheses that: (i) UV radiation activates the previously demonstrated the specific involvement of the Fyn kinase in keratinocyte differentiation NFkB-mediated transcription pathway in LC; and (ii) if correct, UV-induced alterations in control. The lack of Fyn, but not of the closely related Src kinase, is sufficient to determine Langerhans cell function must then be preventable by interfering with the NFkB pathway. For decreased phosphorylation of protein substrates, structural abnormalities as well as decreased this study, we employed the long-term Langerhans cell line XS106, which was established from expression of differentiation-induced proteins in keratinocytes. However, the molecular mechanisms the epidermis of newborn A/J mice and which exhibits features of ‘‘mature’’ Langerhans cells. underlying the specificity of Fyn in keratinocyte differentiation control are still poorly understood. Exposure to unfiltered FS20 sunlamps induced significant activation of NFkB in a time-and To define both specific and overlapping funcions of Fyn versus Src in keratinocytes, we have fluence-dependent fashion, as measured by electrophoretic mobility shift assays (EMSA). Activation generated adenoviral vectors expressing wild-type and mutated forms of both kinases. The effects was detectable with as little as 25 J per m2, and peak responses were observed 1–4 h after exposure of Src and Fyn expression in keratinocytes on phosphorylation of cellular substrates, cell cycle to 200 J per m2. Supershift analyses revealed that p65, p50, c-Rel, and Rel B were all involved control, cytoskeleton organization, cell adhesion and induction of differentiation-specific proteins in this UV-induced NFkB activation. We next transfected XS106 Langerhans cells with a NFkB- will be compared and evaluated. luciferase (Luc) reporter gene; radiation, again, induced significant (Ͼ7-fold) elevation of Luc activity. These results validated our first hypothesis. To block the NFkB pathway, we employed a phosphothioated, double-stranded 20 bp DNA containing the NFkB-binding site. Addition of this NFkB ‘‘decoy’’ nucleotide, but not a scrambled nucleotide control, competitively inhibited the binding of the NFkB probe in an EMSA. Moreover, only the decoy nucleotide inhibited significantly (Ͼ70%) the UV-induced Luc activation, validating its pharmacological activity in XS106 Langerhans cells. UVB radiation induced the secretion of IL-101β, IL-6, and TNF01α by XS106 cells. TNF01α secretion occurred in a dose-dependent fashion up to 200 J per m2, whereas maximal responses for IL-101β and IL-6 were observed at 50 J per m2. Most importantly, addition of the 10 µM NFkB decoy nucleotide resulted in marked inhibition of UVB-induced secretion of each cytokine. For example, the extent of inhibition following 50 J per m2 radiation was 70% (for IL-101β), 67% (IL-6), and 61% (TNF01α). Once again, the scrambled nucleotide had no significant effect. We believe that this is the first report documenting a functional role for the NFkB pathway in UVB-induced cytokine secretion. Our study also forms conceptual and technical bases for the clinical application of the NFkB decoy and its derivatives to avoid the deleterious effects of UV radiation on cutaneous immunity and perhaps to treat other inflammatory skin diseases associated with NFkB activation.

249 250 Immunostimulatory Oligodeoxynucleotides Provide Systemic Therapy For Leishmaniasis Via IL12- A Pool of Disease-Causing Pemphigus Vulgaris IgGs Contains an Autoantibody to Pemphaxin, a and IFNgamma-Dependent Mechanisms Calcium-Dependent Keratinocyte Annexin-like Molecule that can be Upregulated by Methypred- P. S. Walker, A. M. Krieg,* M. C. Udey and J. C. Vogel nisolone Dermatology Branch, NCI, Bethesda, Maryland; *Department of Internal Medicine, University V. Nguyen, A. Ndoye and S. Grando of Iowa, Iowa City, Iowa Department of Dermatology, University of California, Davis, California Resistance to murine leishmaniasis correlates with development of a CD4ϩ predominant Th1 Pemphigus vulgaris (PV) IgGs target pemphaxin (PX), a novel annexin-like molecule that can act immune response. We have demonstrated that immunostimulatory CpG-containing oligodeoxynu- as a cholinergic receptor in human keratinocytes (KC). We investigated the pathophysiologic cleotides (CpG-ODN) can induce a Leishmania-specific Th1 immune response, when CpG-ODN significance of anti-PX antibody in PV. To determine its role in blister development, the PV IgG and freeze-thawed (F/T) Leishmania major are coinjected intradermally into susceptible BALB/C fraction that caused intraepidermal blisters in neonatal mice was preabsorbed with recombinant mice, and protect 40% of animals from subsequent challenge with infectious organisms. In contrast, PX. This eliminated acantholytic activity of PV IgGs which could be restored by adding back the 0% of animals injected with F/T Leishmania organisms and PBS, F/T organisms and control anti-PX antibody eluted from the affinity column. Injection of anti-PX antibody alone did not ODN, or F/T organisms alone are protected. Even more striking protection (65%–95%) is seen cause gross blisters in neonatal mice. The contribution of anti-PX antibody to pemphigus in mice first infected with intact Leishmania organisms and then injected with CpG-ODN, either acantholysis was investigated in the monolayers of human KC treated with the affinity purified at the site of infection or at a remote site. To better understand the mechanisms of these effects, anti-PX PV IgG. Within 120 min of the exposure, KC shed into the culture medium the both CpG-ODN and control ODN were incubated with BALB/c splenocytes, and significantly fragments of their cell membrane carrying desmogleins, as determined by immunofluorescence, more IL-12 (4X) and IFNgamma (8X) were induced by CpG-ODN than control ODN. To and then turned into round acantholytic cells. Thus, binding of PV IgG to PX is essential stage determine if the therapeutic protection provided by CpG-ODN were dependent on IL-12 and of pemphigus acantholysis during which KC cast off their desmosomes into the intercellular space. IFNgamma production, IFNgamma deficient BALB/C mice were treated with either CpG-ODN, The anti-PX PV IgG recognized the 38 and 80 kDa keratinocyte proteins, suggesting that two ODN, or PBS following inoculation with Leishmania. None of these IFNgamma-deficient mice PX molecules are linked covalently in a dimer, which is typical for certain annexins. Since other survived (0/20, 0/20, 0/20, respectively). The role of IL-12 in CpG-ODN protection was assessed annexins (e.g. lipocortin) are upregulated by glucocorticosteroids, producing anti-inflammatory by neutralizing IL-12 in BALB/C mice with intraperitoneal injection of anti-IL12 mAbs, 1 d action, we tested the effects 601α-methylprednisolone (MP) on PX expression. KC were exposed prior to infection with Leishmania and treatment with CpG-ODN. Neutralization of IL-12 for 48 h to 0.25 mM MP or 1.8 mM Ca2ϩ and the cellular proteins were extracted and analyzed completely abolished the therapeutic protection provided by CpG-ODN (0/20 mice surviving). by western blotting using anti-PX PV IgG. As expected, both MP and high Ca2ϩ increased the These results demonstrate that CpG-ODN exert systemic effects via IL-12 and IFNgamma- amounts of PX in KC by 3- and 10-fold, respectively. Since the same dose of MP has been dependent mechanisms and hold considerable promise as both vaccine adjuvants and potential shown to prevent PV IgG-induced acantholysis in skin culture, we propose that glucocorticosteroids therapeutic agents in the prevention and treatment of leishmaniasis. treat pemphigus by stimulating expression of PX in KC. This would counterbalance the deleterious effect of the disease-causing pemphigus antibodies that target PX, leading to shedding of desmosomes by KC and acantholysis.

251 252 Development of a 12-mer Peptide Inhibitor of Langerhans Cell Migration High Doses of Pentoxifylline Accelerate Healing of Venous Ulcers M. Mummert, M. Mohamadzadeh and A. Takashima V. Falanga Department of Dermatology, UT South-western Medical Center, Dallas, Texas Boston University School of Medicine, Department of Dermatology and Skin Surgery, Roger Migration of Langerhans cells from the epidermis into lymph nodes is the initial event leading to Williams Medical Center, Providence, Rhode Island the induction of cellular immune responses to antigens that enter the skin. We have observed Several small studies have shown that systemic administration of pentoxifylline (Trental) may recently that hyaluronan (HA), which is expressed on keratinocytes, mediates hapten-triggered accelerate healing of venous leg ulcers. In the present trail, 15 centers participated in enrolling Langerhans cell migration out of the epidermis. In an attempt to translate this knowledge into a and studying patients with one or more venous ulcers in the outpatient setting. The study was clinically applicable form, we sought to identify peptides that would interfere with the function prospective, randomized, double-blind, and parallel group placebo controlled. All patients received of HA. For this aim, we employed a phage display strategy. Briefly, we started from a phage standardized compression bandaging for treatment of their ulcers and were randomized to receive display library consisting of random 12-mer peptides fused to gIII protein of M13 phage, with 9 11 either pentoxifylline 400 mg, pentoxifylline 800 mg (two 400 mg tablets), or placebo tablets three the complexity of about 10 . We incubated 10 phage clones on plates that had been coated times a day for up to 24 wk. The main outcome measure was time to complete healing of all leg with HA and counter-coated with BSA. After extensive washing to remove unbound phage, only ulcers, using life table analysis. The study was completed as planned in 131 patients. Patients those clones that bound to the HA substrate (but not the polystyrene surface or BSA) were eluted receiving 800 mg three times a day of pentoxifylline healed faster than placebo (p ϭ 0.043, by hyaluronidase treatment. After four rounds of panning, we isolated and sequenced 19 Wilcoxon test). The median time to complete healing was 100, 83, and 71 d for placebo, independent clones. An identical 12-mer peptide motif, termed ‘‘Peptide 1’’, was found in the pentoxifylline 400 mg, and pentoxifylline 800 mg three times a day, respectively. Over half of all majority (13 of 19) of clones, validating the efficacy of our panning. Peptide 1 showed no apparent patients were ulcer free at week 16 (placebo) and at week 12 (both pentoxifylline groups). sequence homology to any of the known ligands of HA or other currently recognized polypeptides. Treatment with pentoxifylline was well tolerated with similar drop-out rates in all three treatment A synthetic peptide containing this motif showed significant binding to HA-coated plates [ELISA] and to the surface of HA-expressing keratinocytes [FACS], and the binding was abrogated groups. In conclusion, treatment with pentoxifylline at a high dose (800 mg three times a day) completely by hyaluronidase treatment. By contrast, none of the control 12-mer peptides showed accelerates healing of venous ulcers. significant binding. To test the in vivo activity, we employed the standard Langerhans cell migration assay, in which BALB/C mice were painted with DNFB on ear skin and then examined for the density of IAϩ Langerhans cells. Two local injections of Peptide 1 (40 µg per ear per injection) at 25 and 1 h before DNFB painting resulted in Ͼ90% inhibition of Langerhans cell migration, whereas control peptides showed no effect. Thus, Peptide 1 is a highly effective inhibitor of Langerhans cell emigration. Because HA, which is also expressed on endothelial cells, mediates homing of pro-inflammatory leukocytes, we next tested the potential of Peptide 1 to block leukocyte immigration into the skin. DNFB-induced ear swelling was inhibited significantly (p Ͻ 0.01) by local injections of Peptide 1, but not by control peptides, in both nonsensitized mice (skin irritant reaction) and DNFB-sensitized mice (allergic reaction). We propose that Peptide 1 and its derivatives represent an entirely new class of anti-inflammatory agents that block leukocyte emigration from, and immigration into, the skin. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 565

253 254 Successful In Vivo Blockade of CD25 (High-Affinity IL-2 Receptor) on T-cells by Administration Fecal and Percutaneous Excretion Rates of 2,3,7,8 TCDD in Two Patients with Severe Intoxication of Humanized Antibody to Psoriasis Patients and Chloracne. Increase of Intestinal Excretion by Oral Administration of Olestra, a Fat Substitute J. Krueger, I. Walters, M. . Miyazawa, P. Gilleaudeau, A. Sherr, S. Light, J. Hakimi and A. Gottlieb A. Geusau, M. Meixner, S. Sandermann, E. Tschachler, G. Stingl, E. Valic, C. Wolf, H. Ru¨diger, Investigative Dermatology, The Rockefeller University, New York, New York; Dermatology, R. Webb, O. Pa¨pke and M. McLachlan UMDNJ-Robert Wood Johnson Medical School, New Brunswick, New Jersey; Protein Design Departments of Dermatology and Internal Medicine IV, University of Vienna Medical School, Labs, Fremont, California; Hoffmann-La Roche, Nutley, New Jersey Vienna, Austria; Procter and Gamble Company, Cincinnati, Ohio; ERGO Forschungsgesellschaft, The IL-2 R 01α-subunit (CD25) interacts with 01β and 01γ chains to form a high-affinity Hamburg, Germany; and Institute of Ostseeforschung, University of Rostock, Warnemu¨nde, receptor that controls T-cell growth. One therapeutic approach to a chronic, T-cell-mediated Germany disease such as psoriasis is to prevent ongoing T-cell activation through blockade of high-affinity In spring 1998, two patients with chloracne were diagnosed at our department. Since the IL-2 receptors. Towards this goal, we conducted a safety and feasibility study of blocking CD25 development of chloracne is the most sensitive marker for a previous exposure to polychlorinated on T-cells of patients with psoriasis vulgaris. A humanized CD25 monoclonal antibody (ZenapaxR) aromatic compounds in man, blood investigations for dioxins and furans were initiated. In both was administered at a 2 mg per kg loading dose followed by four infusions with 1 mg per kg at patients 2,3,7,8 tetrachloro-p-dibenzodioxin (2,3,7,8 TCDD) was detected with a peak level of increasing time intervals over the next 16 wk. CD25 blockade on T-cells was measured by flow 144.000 pg per g blood lipids, the highest ever measured worldwide. The results of scrutinizing cytometry with a murine antibody (clone 2A3) that interacts with the same epitope as ZenapaxR. investigations of the office as well as the patients’ high dioxin blood levels indicate oral intoxication Binding of a murine antibody (clone 7G7) to another epitope was used to quantify total 01α- with 2,3,7,8 TCDD. Due to its lipophilic property 2,3,7,8-TCDD is accumulated in adipose subunit expression. The humanized antibody blocked CD25 completely on circulating T-cells tissue, hardly metabolized, mainly excreted via the intestine and has an estimated half life of 7 y. within 1 h after infusion. Although blocked by ZenapaxR, the 01α-subunit continued to be In this study, we asked whether a diet containing Olestra, a nondigestable, nonabsorbable fat expressed on the cell surface as determined by 7G7 binding. CD25 was also blocked on T-cells substitute could accelerate dioxin excretion. Olestra was fed for 40 d, and TCDD blood levels as in psoriatic skin lesions as assessed by immunohistochemistry in a 1 wk biopsy. As safety-related well as the excretion of this compound via the skin and stool were measured before and during issues, neither total T-cells, nor CD25ϩ T cells, were significantly depleted by administration of the Olestra-rich diet. For the pre-Olestra phase, 2,3,7,8 TCDD was continuously excreted at an this humanized antibody for 16 wk. Nineteen patients tolerated multiple infusions without any even level which was accompanied by a steady linear decline of TCDD blood levels. In the significant drug-related toxicity. Clinical responses ranged from complete clearing of psoriasis to Olestra phase, the intestinal excretion of 2,3,7,8 TCDD was accelerated by the factor 7–10 in no significant disease improvement, but our dosing strategy was inadequate to attain consistent both patients. In contrast, the daily elimination via the skin was only modest representing only CD25 blockade for the duration of the study. This study inicates good clinical tolerability of 1% of total elimination rate and could not be significantly increased by application of petrolatum. relatively high doses of a humanized antibody targeted to activated T-cells and provides impetus The acceleration of the elimination of dioxin by Olestra represents a novel and, hopefully, clinically for additional studies. effective treatment strategy for an otherwise intractable disease.

255 256 A Bilayered Living Skin Construct (Apligraf) Accelerates Complete Closure of Hard-to-Heal Stromal Progenitor Cells Present within Liposuction and Reduction Abdominoplasty Fat for Venous Ulcers Autologous Transfer to Aged Skin V. Falanga and M. Sabolinski* K. Smith, M. Stashower, J. Williams and H. Skelton Boston University School of Medicine, Department of Dermatology and Skin Surgery, Roger National Naval Medical Center, Bethesda, Maryland, Laboratory Corporation of America, Williams Medical Center, Providence, Rhode Island; and *Organogensis, Inc., Canton, Massa- Herndon, Virginia chusetts Autologous fat is used for direct transfer to locally replace fat, as well as for use intradermally in The efficacy of a bilayered, living skin construct (Apligraf) was evaluated in patients (n ϭ 120) the treatment of wrinkles in aged skin. with hard-to-heal venous leg ulcers of greater than 1 y duration. The study was prospective, Liposuction material from three patients and fat from abdominoplasty from five patients was randomized, and controlled. Patients received Apligraf plus compression therapy, or standard processed by homogenization and centrifugation to separate the useful cellular material from the compression alone (active control), and were evaluated for complete wound closure. Treatment blood and cellular debris. The cellular layer was evaluated histologically with routine H&E stains with Apligraf was significantly more effective than active control in the percentage of patients and immunohistochemical including the progenitor cell marker CD34, Bcl-2 and Factor XIIIa. healed by 6 mo (47% vs 19%; p Ͻ 0.005), Fisher’s exact test, 2-tailed) and the median time to The cellular layer showed concentrated spindle cells, some small vascular channels, and a small complete wound closure (p Ͻ 0.005, log-rank test). Analysis with multivariate regression methods, amount of mature fat and stromal material in some sections. CD34 showed diffuse staining of adjusting for factors generally thought to influence healing (i.e. duration, baseline ulcer area, spindle and endothelial cells in all sections with only focal staining of spindle cells with Factor depth, location, fibrinous wound bed, and infection), showed that patients treated with Apligraf XIIIa and light to moderate cytoplasmic staining for Bcl-2. Abdominoplasty fat showed greater were twice as likely to achieve complete wound closure by 6 mo (p Ͻ 0.005), and over 60% CD34 staining of the cellular layer than the liposuction material. more effective in achieving wound closure than active control (p Ͻ 0.01). These data suggest that Processed liposuction and abdominoplasty processed fat may be ideal for the treatment of wrinkles Apligraf is an effective treatment for venous ulcers of greater than 1 y duration. in aged skin because it contains progenitor stromal cells that are present within the fat.

257 258 Molecular Basis of Tobacco Smoke Extract Induced Premature Skin Aging Elongation Factor 1-alpha is a Cytoskeletal-Associated Protein which Functions in Keratinocyte L. Yin, A. Morita and T. Tsuji Signal Transduction Department of Dermatology, Nagoya City University Medical School, Nagoya, Japan A. R. Haake and I. N. Roublevskaia Some epidemiological studies showed the possible association between tobacco smoking and skin Department of Dermatology, University of Rochester School of Medicine and Dentistry, Rochester, aging, but few in vitro studies have directly explored the potential mechanism of tobacco smoke- New York induced premature skin aging. In the present study, we investigated the alteration of collagen, EF-101α is a multifunctional actin-binding protein which controls proliferation and apoptosis. matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in human Previously we showed that EF-101α expression levels and distribution change during skin aging fibroblasts treated with tobacco smoke extract. Human fibroblasts from healthy skin were prepared and keratinocyte passage. Using a sense/antisense strategy we showed that altered levels of EF- and exposed to different concentrations of water-soluble extract from tobacco smoke. Ultraviolet 101α affect the keratinocyteı´s proliferative and apoptotic responses to UV light, retinoids, A1 (340–400)-irradiated human fibroblasts were used as positive control because the mechanism and EGF. Here, we hypothesized that changes in EF-101α levels, isoforms or subcellular of UVA1-mediated gene expression was well investigated. MMP-1, MMP-3, TIMP-1 and TIMP- compartmentalization may control keratinocyte signal transduction in response to extrinsic factors. 3 were analyzed by using reverse trancriptase-polymerase chain reaction followed by the To address this, we have analyzed EF-101α proteins in different subcellular compartments of semiquantitative analyses using NIH Image software. Production of type I and type III collagen fractionated primary keratinocytes by Western Blot prior to and following treatment with EGF (2 was detected by western blotting and newly collagen biosyntheses were assessed by pepsin-resistant nm, 20 nM) or 1.2 mM Ca2ϩ. 2-color fluorescence was used to determine the cellular distribution and successive salt precipitation using 3H-proline incorporation. Our study provided the evidence of EF-101α and its relationship to the actin cytoskeleton in treated and nontreated primary that mRNA expression of MMP-1 and MMP-3 was significantly increased as dose kinetic manner. keratinocytes and EF-101α antisense-expressing HaCaT cells. Our studies show that different EF- The maximum induction was observed at the concentration of 25 µm per ml, whereas the mRNA 101α isoforms are present in the cytosol and cytoskeleton, that rapid subcellular translocation of expression of TIMP-1 and TIMP-3 remained unchanged. Similar observations were also found EF-101α occurs during stimulation of keratinocyte signalling pathways, and that decreased EF- in the UVA1-irradiated human fibroblast. Western blotting of supernatant medium from the same 101α levels render keratinocytes relatively less responsive to extrinsic factors. Changes in EF- treatment revealed that type I and type III collagen was decreased compared with control. Collagen 101α levels or post-translational modifications may contribute to defective signal transduction biosynthesis was significantly reduced by 59% at the concentration of 25 µm per ml. Moreover, characteristic of aged cells. sodium azide (NaN3), L-ascorbic acid and Trolox (a water soluble Vitamin E) prevented the tobacco-induced alteration of matrix metalloproteinases-1 (MMP-1). These observations suggest that the imbalance of connective tissue matrix components might be associated with connective tissue damage, a hallmark of premature aging. The findings further implicated the potential mechanism that singlet oxygen might be important intermediates in the process of tobacco smoke extract-induced as well as UVA-induced skin aging. 566 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

259 260 Interest of a 5% Vitamine C W/O Emulsion in the Treatment of Skin Aging: Effects on Skin Relief Non-Invasive Instrumental Evaluation of the Processes Underlying Skin Maturation, Intrinsic and H. Zahouani, A. Rougier,* P. Creidi,† A. Richard* and P. Humbert† Extrinsic Aging Department of Surface Technologies, Ecole Centrale de Lyon, Lyon, France; *La Roche- C. Cole, J. Pote, G. Payonk, B. Wiegand and J. Gordon Posay; Pharmaceutical Laboratories, La Roche-Posay and Courbevoie, France; †Department of Model and Method Development and Skin Biol Research Ctr, Johnson & Johnson Consumer Dermatology, University Hospital Saint Jacques, Besancon, France Products WorldWide, Skillman, New Jersey In relation to dermatology are the role of vitamine C as reductant and in collagen synthesis. From birth, human skin changes with time, under intrinsic programs resulting in maturation However, only a small amount of work deal with the interest of topical application of vitamine followed by aging and external influences, such as photodamage. Non-invasive instrumental assays C in chronic skin damages in which oxydative stress and collagen synthesis play a determinant were employed in this study to understand changes in skin properties as a function of age. Optical role, such as skin aging. properties were measured using a Minolta Chromameter and a SkinSkan spectrofluorimeter to The present study investigated, in 20 female volunteers (55–60 y) with photoaged skin (low neck), assess skin pigmentation and fluorescent chromophores, respectively. Mechanical properties the effect of a 3-mo daily application of a 5% vitamine C w/o emulsion on changes in (extensibility, elasticity and fatiguability) were evaluated with a Courage & Khazaka Cutometer. topographical, biophysical and mechanical properties of the skin. The study was conducted in Measurements were conducted in early spring on the volar and dorsal forearms of subjects with double blind versus placebo (cream devoided of vitamine C). equivalent numbers of males and females in eight age categories; newborns (2–4 mo), infants (6– The method involved a negative replica of the skin areas before and 3 mo after treatment with 9 mo), toddlers (12–18 mo), children (4–6), teens (13–18), young adults (20–25), adults (30–40) either the 5% vitamine C cream or the placebo. The relief of the replicas was measured using a and seniors (60–70). The data support two patterns: (i) a pattern that was nonlinear with age and threedimensional laser microscope allowing a resolution of 1 µm. The three-dimensional image showed dramatic changes between 3 mo to 4 y, and fewer changes thoughout the adolescent and obtained was then analysed using a new approach of skin surface morphology which allowed to adult periods. These markers included increased yellowness and decreased redness, extensibility, classify skin farrows according to their depth, width and orientation. The furrow families can be tryptophan fluorescence (proliferation) and collagen. These changes were more pronounced on represented as a three-dimentional tree which permited to classify the organisation of skin relief the dorsal forearm and appear to reflect changes due to skin maturation. (ii) Some markers such in three main cathegories, i.e. microrelief (0–10 µm depth), medium (10–20 µm) and deep as skin darkening, elasticity, and elastin fluorescence showed a gradual linear change with time furrows (Ͼ20 µm). throughout life. These kinetics, together with the observation that the rate of change was much Results showed that compared to placebo there more pronounced on the sun exposed dorsal forearm can best be explained by an effect of exists a highly significant increase in the density of skin micro relief as well as a decrease of the continuous sun exposure. The instrumental markers reveal changes specific to skin maturation or deep furrows on the side of the low neck treated with the vitamine C cream. Respectively 70% apparent extrinsic and intrinsic aging. A full understanding of intrinsic aging will require and 74% of the patients showed an improvement of these skin relief parameters. Clinical measurements of sites rarely exposed to sun. examination by the dermatologist as well as self assessment by the patients confirm the improvement of the skin relief [especially of the fine coarse wrinkles (p Ͻ 0.001)]; thus confirming the relevance of the new approach of the measurement of skin surface morphology used in this study. Our data clearly show that topically applied vitamine C can have a beneficial effect in the treatment of skin aging.

261 262 Age Associated Decrease in DNA-Damage Repair: Mechanisms and Implications Age-Dependent Differences in UVA-Induced Oxidative Damage in Human Skin Cells F. Gad, D. A. Goukassian, M. Yaar, M. Eller and B. Gilchrest T. Blatt, R. Keyhani, V. Schreiner, U. Scho¨nrock, S. Gohla, U. Hoppe, D. Schachtschabel* and Department of Dermatology, Boston University School of Medicine, Boston, Massachusetts F. Sta¨b Ultraviolet (UV) irradiation leads to DNA photoproducts in skin that, when not promptly *Clinical Department of Physiological Chemistry, University Marburg; Paul Gerson Unna Research removed, can give rise to mutations and potentially skin cancer. The repair of UV-induced DNA Center, Beiersdorf AG, Hamburg, Germany damage requires recognition and removal of photoproducts and cell cycle arrest during the repair In previous in vivo studies using the UVA-induced ultra-weak photon emission technique we process. Because skin cancer incidence increases sharply with age and indirect measures suggest a found a significant reduction of oxidative stress resistance in elderly skin, which could be improved decrease with age in DNA repair, we wished to determine directly whether there is an age- by topical application of antioxidants (i.e. ubiquinon 10, alpha glucosylrutin). The results of our associated decrease in DNA repair. Human dermal fibroblasts (fb) derived from newborn, young comparative in vitro investigations on primary human fibroblasts and keratinocytes from biopsies adult (25–34 y) and old adult (64–88 y) donors (six donors per age group) were irradiated with of old and young donors demonstrate an age-dependent reduction in the constitutive state of solar simulated light (50 mJ per cm2 as metered at 285 Ϯ 5 nm) and the rate of removal of mitochondrial membrane potential (MMP) and an enhancement in phosphorylation of intracellular photoproducts was examined by slot blot analysis of irradiated DNA at multiple timepoints over tyrosines (PT). However, in the constitutive state of intracellular thiol levels and the phosphorylation 24 h, using appropriate antibodies. There was a significant decrease with aging in the repair rates of intracellular serines and threonines we could not find any age-dependent differences. UVA- of both thymine dimers and (6–4) photoproducts (p Ͻ 0.0001, analysis of covariance). To induced oxidative stress (5–20 J per cm2) reduces the MMP and the intracellular thiol level of determine if this is due to decreased constitutive and/or induced levels of DNA repair and/or cell primary keratinocytes and fibroblasts from young and old donors significantly. At a dose of 20 J cycle arrest, total cellular proteins of sham or UV-irradiated fb from donors of different ages were UVA per cm2 the MMP and the thiol level of fibroblasts from old donors were significantly isolated before and up to 24 h after irradiation. By western blotting, there were age-associated reduced compared with young. The pretreatment of skin cells using antioxidants (i.e. ubiquinon decreases in the constitutive levels of the excision-repair cross complementing 3 (ERCC3) (p Ͻ 10) or o-acetyl-carnitine inhibited significantly the depletion of MMP and intracellular thiols by 0.008), proliferating cell nuclear antigen (PCNA) (p ϭ 0.055) and replication protein A (RPA) UVA-irradiation. In accordance with our results from previous in vivo studies the presented in vitro (p ϭ 0.05) (regression analysis, three donors per age group). However, the most striking difference data confirm that skin cells of old donors are per se more sensitive against oxidative stress, but was observed in xeroderma pigmentosum A (XPA) level that was comparable between newborn can be protected in vitro and in vivo by pretreatment using special actives. and young adult fb but was reduced (p Ͻ 0.014) by 65% Ϯ 12% in old adult fb. Furthermore, within 24 h after irradiation p53 and p21 inductions in old adult fb were consistently lower: ~3- fold of sham-irradiated levels versus ~13-fold (newborn) and ~6-fold (young adult) for p53; and ~2-fold of sham irradiated levels versus ~6-fold (newborn) and ~3-fold (young adult) for p21. To determine if the decreased levels of proteins that participate in DNA repair is controlled at the mRNA level, northern blots were performed. There were significant age-associated reductions in XPA (p Ͻ 0.01) and ERCC3 (p Ͻ 0.0001) mRNAs (SPSS correlation procedure). Our data suggest that the decreased repair of UV-induced DNA damage with aging is due in part to decreased constitutive levels of proteins that participate in the repair process. Furthermore, because XPA acts early during the repair process to recognize the DNA photoproducts, the decreased level of XPA in the elderly may be the rate limiting step for adequate repair of UV-induced DNA damage and hence may be a major contributor for their development of skin cancer.

263 264 Human Desmocollin 1a Expressed in Cultured Mammalian Fibroblast-Like Cells is Bound by Healing of Patients with Bullous Pemphigoid is Correlated with the Loss of Anti-BPAG2 Antibody IgG4 Antibodies in a Pemphigus Foliaceus Serum Subpopulations M. Dmochowski, Z. Nie, C. Kiyokawa and T. Hashimoto C. Yousri, D. Gilbert, P. Lauret, F. Tron and P. Joly Department of Dermatology, Kurume University School of Medicine, Kurume, Japan Service de Dermatologie, Groupe de Recherche en Immunopathologie (Institut Fe´de´ratif de Human desmocollin (Dsc) 1 is an autoantigen in subcorneal pustular dermatosis type of IgA Recherche Multidisciplinaire sur les Peptides, IFR 23), Faculte´ de Me´decine et de Pharmacie, pemphigus. Moreover, Dsc, particularly bovine Dsc, are recognized by IgG antibodies in certain Centre Hospitalier et Universitaire Charles Nicolle, Rouen, France sera of various pemphigus types by immunoblotting. In this study, 10 pemphigus sera were Bullous pemphigoid (BP) is an autoimmune blistering disease characterized by the production of examined by immunofluorescence for IgG antibodies to human Dsc 1a, Dsc 2a and Dsc 3a autoantibodies directed against BPAG1 and BPAG2, 2 proteins of the bazement membrane zone transiently expressed in COS-7 cells and NIH 3T3–3 cells using two different transfection methods. (BMZ). To assess the apparent paradox that there is no correlation between the titers of anti- The lipofectAMINE reagent and superFect reagent were used for transfection of COS-7 cells and BMZ antibodies and the course of the disease, we reevaluate the evolution of anti-BMZ antibodies NIH 3T3–3 cells, respectively. A number of sera with IgG antibodies showed strong background during the course of BP by studying their titers, antigenic specificities and isotypes in paired sera staining with untransfected COS-7 cells and NIH 3T3–3 cells. Using COS-7 cells, a pemphigus of 40 BP patients collected during the active phase of the disease (initial sera) and during remission foliaceus (PF) serum, which did not stain untransfected COS-7 cells, was found to contain IgG (healed sera) after a mean follow-up period of 18.4 mo. The mean immunofluorescence (IF) titers antibodies to Dsc 1a only. This serum showed the same reactivity in studies using NIH 3T3–3 and the immunoblotting profile of anti-BMZ antibodies did not show major differences between cells. Moreover, this PF serum contained IgG4, but not IgG1, antibodies to Dsc 1a expressed in initial and healed sera, when anti whole IgG antibodies were used as the tracer. Comparison of NIH 3T3–3 cells. These results indicate that autoantibody response in PF might be more IgG subclasses of anti-BPAG1 and anti-BPAG2 antibodies in paired sera showed that 78% sera heterogenous than hitherto supposed. had the same anti-BPAG1 antibody IgG subclass pattern, whereas anti-BPAG2 IgG subclasses, especially IgG4 and less frequently IgG1, disappeared from 70% sera from healed patients. Accordingly, the mean anti-BMZ antibody titers of anti-BPAG1 antibody positive sera remained stable during the course of the disease, whereas that of anti-BPAG2 positive sera decreased from seven dilutions between initial and healed sera (p ϭ 0.046). Interestingly, whereas both IgG1 and IgG4 subclasses could be detected by immunoblotting in anti-BPAG2 antibody positive sera, IF titration experiments showed that anti-BPAG2 antibodies mainly correspond to IgG4, whose titers decreased drammatically when patients healed. These data support a major role for anti-BPAG2 IgG4 antibodies in BP patients, that is in accordance with their pathogenic properties in animal models. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 567

265 266 Polymorphic Amino Acid Residues of HLA-DRB1*04 and DRB1*14 Alleles are Involved in Pemphigus Vulgaris IgG Causes Internalization of Clusters of Desmoglein 3, but Not of Autoimmune Responses to Desmoglein 3 (Pemphigus Vulgaris Antigen) Split Desmosomes S. Miyagawa, M. Amagai, H. Niizeki, Y. Yamashina, T. Kaneshige, T. Nishikawa and T. Shirai Y. Kitajima, M. Sato and Y. Aoyama Department of Dermatology, Nara Medical University, Nara; Departments of Dermatology, Keio Department of Dermatol, Gifu University of School of Med, Gifu, Japan University Medical School and National Tokyo Medical Center, Tokyo; and Diagnostic Science We previously showed that desmoglein 3 (Dsg3) was distributed as small clusters on the plasma Department, Shionogi & Co Ltd, Osaka, Japan membrane as well as desmosomes. It is still unclear whether pemphigus vulgaris (PV) IgG splits Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are caused by autoantibodies against the desmosome and the resultant half-desmosome is internalized. We determined whether keratinocyte adhesion molecules. The autoantigens are desmoglein 3 (Dsg3) for PV and desmoglein desmosomes or nondesmosomal Dsg3 clusters were internalized using a human carcinoma 1 (Dsg1) for PF. Recent studies suggest that Dsg1 may also participate in the autoimmune responses cell line, DJM-1. We used a time-lapsed labeling for FITC/Rhodamine(Rod) double-stained of PV patients. To determine possible MHC class II associations with autoantibody responses to immunotluorescence and 5 nm/10 nm gold(G) doublestained immunoelectron microscopy. Cells desmogleins, haplotype and allele distributions, along with molecular polymorphisms of HLA-DR were first treated with PV-IgG for 5 min, washed and labeled in antihuman IgG-FITC or – and -DQ genes were analyzed based on the PCR-RFLP results in 85 Japanese patients with 5nmG antibody-containing medium for 5 min, followed by chasing for 60 min in normal medium pemphigus. The results were as follows: each of 55 PV patients carried at least one allele of HLA- without antibodies. Then, cells were again incubated with PV-IgG medium for 5 min, washed DRB1*04 and DRB1*14 subtypes, with significant increases of HLA-DRB1*0406/DQA1*0301/ and stained with antihuman IgG-Rod or – 10nmG antibodies for 5 min. By this method, FITC DQB1*0302, DRB1*1401/DQA1*0104/DQB1*0502, DRB1*1401/DQA1*0104/ and 5nmG particles show the fate of Dsg3-PV-1gG complexes formed during the first 5 min. DQB1*05031, DRB1*1405/DQA1*0104/DQB1*05031 and DRB1*1406/DQA1*0503/ Rod and 10nmG show new Dsg3 molecules expressed on the cell surface during the chase. Labels DQB1*0301 haplotypes (p Ͻ 0.0001 for each haplotype) compared to normal controls. The can not penetrate into the cells because labeling was performed on the living cells, so that Rod HLA-DRB1*04 and DRB1*14 alleles carried by PV patients shared hydrophobic amino acid and 10nmG must indicate newly expressed or continuously existing Dsg3 on the cell surface. residues Phe26, Leu67 and Val86, as well as hydrophilic amino acid residues at positions 70 and 71 After 60 min-chasing, many endosomes containing only 5nmG were found in the cell, and most on the DRB1 beta chain; HLA-DR/DQ distributions did not differ among PV patients according cell-surface labelings were 10nmG-clusters. Many dots of FITC but not of Rod were seen in the to the presence or absence of anti-Dsg1 coexisting with anti-Dsg3; and 30 PF patients, all cell. These results indicate that PV-IgG-Dsg3 complexes were internalized. Desmosomes were producing autoantibodies only to Dsg1, showed more diverse HLA-DR/DQ distributions, sharing labeled with both 5nmG and 10nmG, indicating they continuously exist without splitting. Half- hydrophobic amino acid residues at positions 26 and 67, as well as hydrophilic amino acid residues desmosome-like structures were labeled with only 10nmG but not with 5nmG suggesting that at positions 70 and 71, of the DRB1 chain. These findings suggest that autoantibody responses to they were new products during the last 60 min, but not of split desmosomes. These suggest that desmogleins might be regulated by amino acid residues at positions 26, 67, 70, 71, and 86 at internalization of nondesmosomal Dsg3, but not of split desmosomes, is a primary trigger for PV- peptide binding sites of HLA-DRB1 molecules, and that autoimmune responses to Dsg3 might IgG effects on keratinocytes. be more strictly regulated by specific amino acid residues at these positions on the HLA-DRB1 chain than responses to Dsg1.

267 268 Mucosal Morbidity in Patients with Epidermolysis Bullosa Acquisita (EBA) Predominant IgG4 Subclass is Responsible for the Initial False Negative IgG for Bullous Pemphigoid M. Luke, T. Darling, R. Hsu, R. Summers, J. Smith, B. Solomon, G. Thomas and K. Yancey Skin Direct Immunofluorescent Study Dermatology Branch, NCI; Department of Diagnostic Radiology; Laboratory of Immunology, M. Seraly, H. Thong, J. Deng, R. Draviam and J. Abernethy NEI; Department of Rehabilitation Medicine; and Head and Neck Surgery Branch, Tumor Department of Dermatology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania Biology Section, NIDCD; National Institutes of Health, Bethesda, Maryland Bullous pemphigoid (BP) is characterized by the presence of IgG and C3 along BMZ of skin. EBA is an acquired inflammatory and/or dermolytic subepidermal blistering disease characterized Positive C3, but negative IgG DIF has been noticed in some BP patients. Ten patients known to by IgG autoantibodies to type VII collagen. To characterize mucosal involvement and its have early stage of BP are included in this study. The criteria for diagnosis of BP include: clinical complications in EBA, four patients were evaluated by a multidisciplinary team of care providers manifestations including urticarial plaques, blisters and erosion of skin; histopathological changes over 1–5 y of continuous study. All patients had features of EBA as well as IgG autoantibodies (infiltration of eosinophils and neutrophils, with/or without dermoepidermal separation), thera- that either immunoblotted type VII collagen in vitro or bound anchoring fibrils in vivo. Patients peutic response to systemic corticosteroid, and at least positive C3 DIF staining along BMZ of were evaluated clinically as well as by slit lamp examinations, endoscopies, CT scans, and skin. Frozen sections of skin specimens from these 10 patients were subjected to IgG DIF, IgG fluorographic swallowing studies. Spiral CT scans for virtual endoscopy were used for the IIF, IgG subclass IIF and light chain IIF. IgG conjugates used in IgG DIF were screened for their nontraumatic evaluation of two patients with respiratory compromise. Involvement of five or reactivity to IgG subclasses using ELISA. All 10 specimens showed negative IgG DIF, but seven more mucosal sites was documented in all patients; every patient had lesions in their mouth, nose, of them were positive in IgG IIF. Most strikingly, all 10 specimens showed positive linear IgG4 conjunctiva, pharynx, and larynx. Complications included ankyloglossia, periodontal disease, staining along BMZ. There was no IgG light chain restriction. Two IgG conjugates, which were scarring of nasal mucosa, symblephara, obstruction of nasolacrimal ducts, deformation of the used in our laboratory, were analyzed for their specificity to IgG subclasses in ELISA. Both epiglottis, impaired phonation, dysphagia, esophageal strictures, and supraglottic stenosis requiring conjugates showed higher reactivity to IgG1, IgG3 and to a lesser degree to IgG2 and IgG4. emergent tracheostomy. EBA may extensively (or predominantly) affect mucosal epithelia in a These results suggest the following factors contributing to the negative IgG DIF in bullous manner that resembles cicatricial pemphigoid. Mucosal disease in these patients is often subclinical, pemphigoid: (i) subthreshold IgG in skin specimens, (ii) limited reactivity of IgG conjugate to can lead to serious complications, and is best managed using a multidisciplinary approach. IgG4 subclass, (iii) sensitivity of test (DIF versus IIF). This study also strongly indicates that IIF for IgG and/or IgG subclass is useful for bullous pemphigoid patients with negative IgG DIF, but positive C3 DIF.

269 270 IgA Anti-LABD97 Antibodies in Linear IgA Bullous Dermatosis Are Heterogeneous Clinical and Immunologic Features of Paraneoplastic Pemphigus C. Egan, T. Taylor, L. Meyer, M. Petersen and J. Zone H. C. Nousari, A. Kimyai-Asadi and G. J. Anhalt Department of Dermatology, University of Utah, Salt Lake City, Utah Department of Dermatology, Johns Hopkins Medical Institutions, Baltimore, Maryland Linear IgA bullous dermatosis (LABD) is a rare acquired subepidermal blistering disease of the Paraneoplastic pemphigus (PNP) is an autoimmune blistering disorder associated with malignancy. skin. We have previously described a 97-kDa BMZ protein to which circulating IgA antibodies We describe clinical, histologic, and immunologic features of 123 patients with PNP. The mean in LABD patients’ sera bind termed LABD97. We have also shown that this protein shares age of onset was 53 y and two-thirds of patients were male. PNP usually developed after a tumor structural homology with bullous pemphigoid antigen 2 (BPAg2). Previous studies, using wad diagnosed, and the most common associated malignancies include non-Hodgkin’s lymphoma, immunofluorescent techniques, have suggested that the IgA response in LABD is restricted to the chronic lymphocytic leukemia, Castleman’s disease, thymoma, sarcomas, and Waldenstrom’s IgA1 subclass. We studied the IgA antibody subclasses in the sera of six patients that contained macroglobulinemia. Castleman’s disease is the predominant tumor in patients under age 30. All circulating IgA antibodies reactive with LABD97. The methods used included direct and indirect patients had oral mucosal lesions, half had conjunctivitis, and a third had genital involvement. immunofluorescence and western immunoblot. IgA1 and IgA2 antibody subclasses differ by only 95% of patients has cutaneous involvement, most commonly characterized by blisters, erosions, a small number of amino acids, with the majority of the structural difference occurring at the and/or lichenoid lesions. Respiratory symptoms were present in a quarter of patients, and hinge region of the molecule. Therefore it is important to demonstrate that monoclonal antibodies autoantibody deposition on the respiratory tract was confirmed by direct immunofluorescence in that are specific for each subclass do not exhibit antigen cross-reactivity. We demonstrated that five patients. The most common histologic features were acantholysis, lichenoid dermal infiltrates, the antigen specificity of the anti-IgA subclass antibodies used did not cross react by slot basal vacuolization, and dyskeratosis. Direct immunofluorescence most commonly revealed IgG immunoblot. All six patients had IgA1 anti-LABD97 antibodies. Surprisingly, two of these also and C3 deposition on epithelial cell surfaces and/or the basement membrane zone. Indirect had anti-LABD97 IgA2 antibodies and one had secretory component containing anti-LABD97 immunofluorescence showed circulating antibodies binding stratified squamous and transitional IgA antibodies. We conclude that the predominant IgA antibody subclass reactive with LABD97 epithelia, liver desmosomes, and/or myocardial intercalated discs. Immunoprecipitation revealed in LABD is IgA1, although there is heterogeneity of this response with IgA2 being involved in a antibodies to desmoplakins, BPAgl, envoplakin, periplakin, and/or an unidentified 170 kDa minority of cases. antigen. Surgical treatment of Castleman’s disease was the only tumor therapy that effectively controlled PNP. The majority of patients died within a year of PNP onset, most commonly due to respiratory failure or sepsis. 568 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

271 272 Laminin 01α3-Specific Autoantibodies in Patients with Anti-Epiligrin Cicatricial Pemphigoid Analysis of Type VII Collagen Gene (COL7A1) Mutation in Three Japanese Cases of Dystrophic (AECP) Recognize the G Domain of this Polypeptide Epidermolysis Bullosa (DEB) Z. Lazarova, C. Yee, J. Lazar and K. Yancey M. Mayama, K. Tamai, K. Fukai,* T. Nakagawa,† T. Ishikawa,‡ A. Kon, D. Sawamura, K. Dermatology Branch, NCI, NIH, Bethesda, Maryland; and VAMC Research Center, Baltimore, Hanada and I. Hashimoto Maryland Department of Dermatology, Hirosaki University School of Medicine, Hirosaki, Japan; *Osaka AECP is a mucosal-predominant subepithelial blistering disease characterized by IgG antibasement City University School of Medicine, Osaka, Japan; †Kagawa Medical College, Miki, Kagawa, membrane autoantibodies to laminin 5 (01α301β301γ2). Immunoblot studies found that IgG from Japan; and ‡Omagari General Hospital, Omagari, Japan 24 AECP patients bound 01α3(nϭ21), 01α3 and 01γ2(nϭ2), or 01β3 and 01γ2(nϭ1) DEB is a heritable mechanobullous skin disease derived from mutations in the COL7A1. In this subunits of laminin 5. Given the dominance of reactivity to laminin 01α3, sera from 10 study, we examined COL7A1 mutation in three families of different phenotypes, localized representative patients with such autoantibodies were tested against prokaryotic recombinants of dominant DEB, mitis recessive DEB (DDEB), and Hallopeau-Siemens recessive DEB (HS- this polypeptide in epitope mapping studies. Overlapping cDNAs spanning the full length of RDEB), to examine genotype-phenotype correlation. To search COL7A1 mutations, genomic laminin 01α3 were generated by PCR, directionally cloned into pGEX-4T-3, and expressed as DNA from peripheral blood of each cases were used as a template for PCR of all COL7A1 exons, glutathione-S-transferase (GST)-fusion proteins of appropriate size and immunoreactivity. Two followed by heteroduplex analysis and nucleotide sequencing. Identified COL7A1 mutations of different sets of fusion proteins (analyzed with sera from three and seven patients) provided three families are as follows. Case 1, 8068del17ins2; Case 2, E2857X/S1373 L; Case 3, G2395D/ identical results, namely that sera from nine of 10 AECP patients immunoblotted fusion proteins 154delG. Those mutations have not been reported except E2857X, which is known as Japanese corresponding to the carboxyl-terminal G domain of laminin 01α3. One patient (and normal recurrent mutation. 8068del17ins2 is the first reported insertion/deletion mutation resulting in human serum, control) showed no reactivity to any recombinant; no sera bound recombinant DDEB. Type VII collagen alterations in case 2, caused by combination of PTC in NC-2 domain GST. Immunoadsorption of sera from two patients with fusion proteins corresponding to the G and missense mutation in triple helical domain, result in mild phenotype, while those of case 3, domain substantially reduced their basement membrane autoantibody titers (e.g. from 100 to 5 in caused by GS and PTC in NC-1, result in severe form of RDEB. These findings will provides serum exposed to GST or G domain fusion proteins, respectively). These studies show that further understanding for genotype-phenotype correlation of DEB. laminin 01α3-specific IgG autoantibodies in patients with AECP recognize the portion of laminin 5 thought to play a key role in promoting keratinocyte adhesion to epidermal basement membrane. These findings further our understanding of AECP pathophysiology and laminin 5 biology.

273 274 The Clinical Significance of Autoantibodies to Desmoglein 1 in 78 Cases of Pemphigus Vulgaris Autoantibodies Against Type XVII Collagen (BP180, BPAG2) Define a Spontaneously Arising K. E. Harman, M. J. Gratian, B. S. Bhogal, S. J. Challecombe and M. Black Porcine Model of Bullous Pemphigoid St John’s Institute of Dermatology, St.Thomas’ Hospital and the Department of Oral Medicine T. Olivry, M. Mirsky, W. Singleton, S. Dunston, A. Borrillo, L. Xu and L. Chan and Oral Immunology, Guy’s Hospital, London, UK North Carolina State University College of Veterinary Medicine, Raleigh, North Carolina; Pemphigus vulgaris (PV) is characterised by autoantibodies to desmoglein 3 (Dsg3) but many cases Rhoˆne-Poulenc Rorer, Collegeville, Pennsylvania; and North-western University School of have additional autoantibodies to desmoglein 1 (Dsg1), the major antigen in pemphigus foliaceus. Medicine, Chicago, Illinois We have studied the clinical significance and time course of this finding in 78 patients with PV Cutaneous blistering arose spontaneously in multiple adult Yucatan minipigs. Skin lesions consisted diagnosed on the basis of clinical, histological, and direct/indirect immunofluorescence findings. of turgid, isolated or clustered vesicles that occasionally evolved from erythematous and pruritic IgG antibodies to the ectodomain of Dsg1 and Dsg3 were detected by ELISA. One serum sample macules and patches. Histopathological examination revealed subepidermal vesicles rich in intact from each patient was analyzed, ideally taken prior to commencing therapy but otherwise when and degranulated eosinophils. Antigen mapping and/or transmission electron microscopy confirmed they had active disease. Multiple samples were analyzed in 43 patients. that dermoepidermal separation invariably took place in the lamina lucida of the epidermal Sixty-two per cent of PV patients had antibodies to Dsg1 in addition to Dsg3. This finding basement membrane zone. Direct immunofluorescence (IF) revealed the presence of IgG deposited strongly predicted the presence of mucosal and cutaneous ulceration (98%) and was a very unlikely linearly at the dermoepidermal junction in all skin specimens examined. Indirect IF testing finding in patients with purely mucosal PV (2%). Sixty-three per cent of patients with only Dsg3 confirmed the existence, in almost all affected pigs’ sera, of circulating basement membrane- antibodies developed skin lesions but these were usually minor. Extensive, life-threatening specific IgG autoantibodies (titers 1:100–1:500). Using uncleaved and salt-split lip substrates, the cutaneous ulceration was seen only in patients with high Dsg1 and Dsg3 ELISA values. autoantibodies were shown to target antigens situated not only at the basal, but also at the lateral In those subjects who had not commenced treatment when serum was taken, the mean period of and apical aspects of stratum basale keratinocytes. ELISA were performed with synthetic peptides untreated symptoms was shorter in these with additional Dsg1 autoantibodies (5 cf. 22.5 mo). spanning the entire NC16A ectodomain of human BP180. Half of the pig sera exhibited high Analysis of sequential samples showed that Dsg1 autoantibodies were usually present at diagnosis immunoreactivity against NC16A peptides. This novel porcine acquired blistering dermatosis and did not appear for the first time later during the disease. However, immunosuppressive could be proposed therefore as a spontaneous model to study the immunopathogenesis of BP treatment in some cases resulted in a transient or permanent fall in Dsg1 autoantibody levels whilst in humans. Dsg3 antibodies remained detectable by ELISA. In summary, Dsg1 autoantibodies predict the presence of cutaneous disease in PV and a more severe disease phenotype. They are usually detected early in the course of disease and do not seem to be related to external factors such as the delayed use of treatment.

275 276 Paraneoplastic Pemphigus: a Combined Humoral and Cellular Autoimmune Disease Horizontal Averaging in Image Processing for Measuring Area Involved in Pemphigus Patients V. Nguyen, K. Bassler, A. Ndoye, R. Webber,* L. Shultz,† B. Ruben and S. Grando M. Tanaka,*† S. Kobayashi,* S. Gaskell,† C. Edwards† and R. Marks† Department of Dermatology, University of California, Davis, California; *Research & Diagnostic *Department of Dermatology, Keio University School of Medicine, Tokyo, Japan; and †University Antibodies, Richmond, California; and †The Jackson Laboratory, Bar Harbor, Maine of Wales College of Medicine, Cardiff, UK The pathophysiology of paraneoplastic pemphigus (PNP) – an autoimmune disease with character- The aim of the study is to measure the area involved in patients with pemphigus using image istic clinical, histological and immunological features – is a subject of intense research. It has been processing and image analysis. We have previously reported several c-programs of image processing proposed that PNP patients develop acantholytic antibodies targeting desmoglein (Dsg) 3. PNP, for shade correction that enables automatic measurement of the area involved in psoriatic patients. however, may lack acantholysis. The satellite cell necrosis, vacuolar-interface changes and lichenoid The method have been shown to be useful for measuring of lesions on the trunk in patients with infiltrate, found in PNP lesions, suggested a role for cellular cytotoxic reactions. We tested the guttate type psoriasis. In the present study, we further applied the method to measure skin lesions contribution of cell-mediated autoimmunity by staining PNP lesions for inducible nitric oxide in patients with pemphigus. Patient images were read and stored as image raw data with pixels of synthase (iNOS), which is expressed by both the effector and target cells engaged in cellular 720 by 560. The programs were run with images and the processed images were analyzed with cytotoxic reactions. As expected, both the infiltrating mononuclear cells (MNC) and keratinocytes an image analyzer, Image Pro Plus. The processed images were compared with the original images (KC) stained for iNOS. Since PNP antibodies also reacted with KC, an antibody-dependent and shown to correspond to most of the lesions, which included blisters, erosions and erythemata. cellular cytotoxicity might provide a mechanism for epidermal damage in PNP. We therefore It was more difficult in pemphigus than in psoriasis, because the lesions in the former consist of asked if passive transfer of PNP IgGs to neonatal mice can recruit autoreactive murine MNC to new and old skin lesions and of more polymorphous lesions. Although there still are some self epidermis. The PNP IgGs that precipitated KC proteins of 180, 190, 210, 230, and 250 kDa difficulties in detecting polymorphous lesions, this method would be a useful and powerful tool (and uniquely a 40-kDa band) and stained squamous and transitional epithelial substrates were when considering the statistical relationship, for example, between severity and ELISA titers for injected i.p. at 20 mg per kg per g body weight to both the Dsg 3 positive BALB/C and the Dsg Desmogleins 1 and 3. 3 negative Dsg3null mice. The pups of both strains produced mild erythema and gross skin blisters 18–24 h after injection. Lichenoid subepidermal mononuclear infiltrates were found subjacent to the areas of epidermal clefting, which was caused by disintegration of the basal layer with basal KC separating from both the basal membrane and the adjacent KC. Necrotic KC were admixed with MNC, and both cell types expressed iNOS. PNP IgGs were found in the intercellular epidermal spaces and also at desmosome-free areas of separated basal KC. We conclude that the pathophysiology of PNP involves both the humoral and the cellular autoimmunity effectors, with autoantibodies playing a pivotal role, and that keratinocyte cell membrane antigens other than Dsg 3 are targeted by disease-causing PNP antibodies. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 569

277 278 Autoantibodies from Patients with Cicatricial Pemphigoid Shows Diversity of Reactivity to The Effects of Topical and Oral d-alpha-Tocopherol on Pigmentation and Skin Cancer Induced Basement Membrane Zone Proteins by Ultraviolet Irradiation C. Matsui, T. Muramatsu and M. Morohashi K. Burke, J. Commisso, G. Combs, Jr, E. Gross, C. Keen and R. Nakamura Department of Dermatology, Toyama Medical and Pharmaceutical University, Toyama, Japan; Department of Medicine, Cabrini Medical Center, New York, New York; Department of Poultry Department of Dermatology, Nara Prefectural Medical University, Nara, Japan and Avian Sciences, Cornell University Ithaca New York; Department of Dermatology, Newington Cicatricial pemphigoid (CP) is autoimmune bullous disease that primarily affects mucosal tissues. Veterans Administration Medical Center Newington, Conneticut; Department of Nutrition, Recent studies have identified CP patients who have IgG autoantibodies that recognize several University of California Davis, California; Department of Pathology, Scripps Clinic and Research basement membrane proteins, BP180, epiligrin identical to laminin5 (Lam5), and so on. To further Foundation, La Jolla, California understand the pathophysiology of blister formation in these patients, we have sought to identify The purpose of the study was to determine whether supplementation with oral and/or topical d- the specific epitope(s) targeted by their auto antibodies. Immunoprecipitation studies with alpha-tocopherol (vitamin E) and/or topical d-alpha-tocopheryl succinate (vitamin E succinate) keratinocyte show that sera from these patients can be classified into three types, (i) react to BP180 can reduce incidence of acute and/or chronic damage to the skin (i.e. sunburn and pigmentation (BP), (ii) react to Lam5, (iii) react to both of BP and Lam5. Using two types of sera that react to and/or skin cancer, respectively) induced by ultraviolet (UV) irradiation in mice. Groups of 20 BP, we investigated CP-reactive site of BP protein in immunoblotting analysis. Bacterial fusion Skh:2 female hairless pigmented mice were treated with 1. lotion vehicle, 2. 5% vitamin E lotion, proteins that contain noncollagenous (NC) 16A domain and carboxy terminal domain of BP180 3. 5% vitamin E succinate lotion, or 4. lotion vehicle and oral vitamin E. Within each group, 15 were used as substrate. Most of anti BP sera showed reactivity to carboxy terminal domain and mice were exposed to 0.24 J per cm2 of UVB irradiation three times per week. Vitamin E most of anti-BP ϩ anti-Lam5 sera reacted to NC16A domain of BP. All of anti-Lam5 serum concentrations of skin, liver and adipose tissue were measured. Skin pigmentation was scored, and react to 01α3 chain of Lam5 only. The epitope mapping using bacterial fusion protein of 01α3 the total number of clinically detectable skin tumors per animal was counted weekly. Results subunit is now in progress. Furthermore, patients that have two kinds of antibody tend to present showed that skin and liver concentrations of vitamin E were increased after topical application. more serious clinical feature. So-called epitope spreading might have close relationship to severity Mice treated with each form of vitamin E showed no signs of toxicity and had significantly less of CP. acute and chronic skin damage induced by UV, as indicated by reduced inflammation and pigmentation and by later onset and lesser incidence of skin cancer.

279 280 Adult Human Skin Grafted to Severe Combined Immunodeficiency Mice for UVA and UVB Reduced Expression of an Actin Binding Protein, Calponin h 1 (CNhl), in Blood Vessels of Carcinogenesis Studies Human Malignant Skin Tumors C. Berking, N. Black, R. Binder* and M. Herlyn Y. Koganehira, T. Ehara, M. Takeoka, T. Saida and S. Taniguchi The Wistar Institute, Philadelphia, Pennsylvania; *Procter & Gamble Co., Miami Valley Laborat- Department of Dermatology, Department of Pathology, Department of Molecular Oncology and ories, Cincinnati, Ohio Angiology, Shinshu University School of Medicine, Matsumoto, Japan Our laboratory has previously demonstrated the development of human squamous cell carcinomas We previously observed the decrease of smooth muscle 01α-actin in malignant tumor vessels, and (SCC) and melanocytic lesions including melanoma after a combined treatment of DMBA and suggested that growth factors, such as PDGF, secreted by malignant cells suppressed the expression UVB. These studies were done with foreskin from newborns. However, the skin type of the of smooth muscle 01α-actin. CNhl is an actin binding protein, which is largely expressed in newborn donors was difficult to assess at the time of grafting leading in approximately 40% of all smooth muscle cells, stabilizes actin filaments and increases cell adhesion, and negatively regulates cases to the inclusion of skin type IV and V donors. To better select skin from individuals of skin cellular growth and motility. The expression of CNh1 is known to be also down-regulated by type I and II who have a high susceptibility for SCC or melanoma, we have grafted full-thickness PDGF To examine the expression of CNh1 in tumor vessels, we immunologically stained various skin from adults. To achieve a 70%–80% take rate, we had to modify the grafting technique. types of skin tumors with anti-CNh1 antibody. We observed the weaker expression of CNhl in Seventy-three skin specimen from abdomen (12 donors) and 40 from breast (10 donors) were the malignant tumor tissues than in the surrounding normal tissues. Furthermore, the decreased grafted onto 113 SCID mice. The grafts (1.5 01ϫ 1.5 01ϫ 0.1 cm) were applied on the sides of staining with anti-CNhl antibody was seen even in the blood vessels where smooth muscle 01α- the animals leaving the muscle panniculus carnosus intact and were held in place by suturing at actin was detected. Thus, CNhl in tumor vessels is more sensitively suppressed than smooth four sites or by bandaging only. After an observation period of 4–8 wk, grafts were histologically muscle 01α-actin. Such alteration in the expression of actin-related molecules in tumor vessels evaluated by H&E staining, immunohistochemistry for Ki-67, S-100 and involucrin, and by could be a good marker of malignancy, and blood vessels exhibiting low expression of CNhl may nuclear Hoechst dye. Morphologically human-like epidermis but formed by mouse keratinocytes have structural fragility advantageous to metastasis of malignant tumor cells. was detected in 12% of grafts. Hyperpigmentation as well as loss of pigmentation were noticed in some grafts. Scaling and crust development were observed after 2–3 wk followed by healing to clinically normal skin after 5–8 wk. Histology revealed acanthosis of the epidermis. The presurgical preparation of donor tissue, the grafting method itself and the postsurgical bandaging technique were most important for consistent take rates. These preliminary investigations demonstrate our ability to use skin from adult donors with high susceptibility for melanoma for grafting and exposure to UVA and UVB in order to investigate the pathogenesis of human melanoma in this unique model.

281 282 Distinct p53 Gene Mutations in Korean Bowen’s Disease Neurotrophin (NT)-4 Inhibits Growth of Malignant Melanoma Cells H. Lee, J. Kim, S. Oh, E. Seo, W. Park, J. Lee and J. Kim M. Grewe, K. Vogelsang, K. Karmann and J. Krutmann Department of Dermatology and Department of Clinical Pathology, St Paul’s Hospital and Clinical and Experimental Photoderm, Department of Dermatology, Du¨sseldorf, Germany Department of Pathology, Catholic University of Korea, Seoul, Korea Neurotrophins are potent modulators of growth and differentiation of neural crest derived cells We studied the role of UV radiation and possible racial differences in Bowen’s disease in Korean such as nerve cells and melanocytes. Human epidermal keratinocytes have been shown to synthesize by analyzing p53 gene mutations. We performed immunohistochemical staining in 17 samples large amounts of biologically active NT-4, especially under the inflammatory conditions of atopic from 12 patients with Bowen’s disease. Point mutations in p53 gene were studied by single-strand dermatitis. Additionally, recent studies indicated that atopic dermititis patients have significantly conformation polymorphism analysis after amplification of p53 exons 5–8. Loss of heterozygosity decreased numbers of melanocytic nevi. We therefore speculated that NT-4 as a highly expressed (LOH) was also studied by polymerase chain reaction with TP53 (17p13) microsatellite marker. skin derived factor in atopic eczema might influence growth of melanocytic cells. In order to Immunohistochemical staining was positive in 50% (six of 12). Fourteen missense mutations were answer this question, the influence of NT-4 on four different melanocytic cell lines, which have found in 50% (six of 12) and 11 were clustered in exon 5. Seven mutations in five patients were been established from cutaneous, lymphonodular and hematogenic metastases, was assessed. C:G to T:A transitions at dipyrimidine sites. Multiple mutations were observed in two patients Melanoma cells were kept under ‘‘starving’’ conditions for 24 h, and were then incubated in the with long duration of Bowen’s disease. A random discordant pattern of p53 mutation was observed presence of different concentrations of recombinant human (rh)NT-4. After 16 h, melanoma cell in distantly separate tumors in five patients. TP53-LOH was detected in 30% (three of nine). number was assessed by cell count, WST-1, and by BrdU incorporation assays. As measured by Multiple mutations in a lesion was a new finding observed in Bowen’s disease and suggest that these three methods, all four melanoma cell lines showed a NT-4 dose-dependent decrease of cell additional mutations may occur in tumor cells containing mutant p53 for long duration. Multiple number by 30%–60% at an optimal rhNT-4 concentration between 50 and 100 µg per ml. FACS- mutation does not seem to predict the development of invasive SCC or be related with tumor analysis of the Fas/Fas ligand system of the melanoma cells did not show any difference between size. Random discordant mutations indicate independent origin of multiple Bowen’s disease. NT-4 treated or untreated cells, indicating that the differences in cell number after NT-4 treatment were not caused by increased apoptosis. Furthermore, close microscopic inspection of cell cultures as well as trypan blue exclusion assays gave no hints for higher lethality in NT-4 treated melanoma cell cultures. Thus, decrease of cell numbers might most likely be explained by influences of NT- 4 on melanoma cell cycle, e.g. by causing cell cycle arrest. In summary, evidence is provided that a human skin derived factor is able to negatively regulate growth of melanocytic cells. This observation might provide new tools for adjuvant melanoma therapy, or for support of chemotherapy by synchronization of melanoma cells. 570 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

283 284 Retinoid Metabolism Pathways in Human Skin-Squamous Cell Carcinoma Cell Line SCC13 The All-Trans-Retinoic Acid-4-Hydroxylase (CYP26) is Inducible in Human-Skin Squamous Cell T. C. Roos, H. F. Merk and F. K. Jugert Carcinoma Cell Line SCC12 but not in SCC13 Department of Dermatology, University Clinic of the RWTH Aachen, Germany F. K. Jugert, H. F. Merk and T. C. Roos Vitamin A and its derivatives (retinoids) are essential for the regulation of a variety of life processes, Department of Dermatology, University Clinic of the RWTH Aachen, Germany such as vision, reproduction, embryonic development, and the regulation of cell growth and Retinoids are essential for regulation of epithelial growth and differentiation. Their activity is differentiation. There is also considerable evidence that retinoids are effective in the treatment of mediated through interactions with retinoic acid and retinoid x receptors (RAR and RXR). All- hyperproliferative, premalignant and inflammative skin diseases (e.g. psoriasis, Darier’s disease, trans-retinoic acid (atRA) is the ligand for RAR and therefore a key regulator of RAR-activity. actinic keratoses and acne). Although profound progress has been achieved with regard to the The degradation of atRA by 4-hydroxylation seems to be an important step in the regulation of action of retinoids on the molecular level, the metabolic pathways of retinoids in normal and retinoid receptor activity. Recently, a human cytochrome P450 (hP450RAI, CYP26) specific for malignant tissues are largely unclear. Since stable intracellular retinoid-levels appear to be essential the 4-hydroxylation of atRA has been identified. In a previous study we demonstrated that the for optimal retinoid receptor function, irregularities in the pathway of retinoids may have profound CYP26 is inducible by its substrate atRA in human dermal fibroblasts suggesting that this enzyme effects here. The cascade of retinoid activation is driven by enzymes of the alcohol dehydrogenase plays an essential role in the regulation of retinoid activity in the skin and that CYP-26-activity family (ADH), short chain dehydrogenases (SDH), aldehyd dehydrogenases (RalDH), and several may be regulated via a retinoic acid response element (RARE)-feedback loop by its substrate enzymes of the P450 isoenzyme superfamily (CYPs). The inactivation of all-trans-retinoic acid has atRA which has been characterized in the promoter region of this enzyme. been shown to be mediated by a novel CYP26. Using human squamous cell carcinoma cell line In the present study we investigated the CYP26 expression and inducibility in human epidermal 13 (SCC13) derived from facial skin, we investigated the pathways of retinoid metabolization and squamous cell carcinoma cell lines SCC12 and SCC13, in vitro. Using reverse phase HPLC for isomerization on the biochemical level by reverse phase-high performance liquid chromatography the determination of retinoid metabolites and reverse transcriptase PCR to measure CYP26- (RP-HPLC), and on the molecular level by rt-PCR to estimate the expression-levels of several RNA-expression after treatment of the cells with 10–5 M atRA we found a CYP26-induction in retinoid-related RNAs, such as RolDH, RalDH, SDH, cellular retinoic acid binding proteins SCC12 cell line accompanied by the generation of 4-hydroxylated atRA-metabolites, whereas in (CRABP-I/-II), CYP26, retinoic acid receptors (RARs) and retinoid 01ϫ receptors (RXRs). SCC13 cells neither CYP26-expression nor the production of polar atRA-metabolites were SCC13 cells were incubated with either 10–5 M all-trans-retinol (ROL), all-trans-retinal (RAL), traceable after 0, 24, 48, 72, 96, or 120 h, respectively. all-trans-retinoic acid (atRA), 9-cis-retinoic acid (9RA), or 13-cis-retinoic acid (13RA). Specimens Since the CYP26 is also not inducible in human epidermal keratinocytes, in vitro, our results may for RP-HPLC and rt-PCR were obtained after 24, 48, 72, 96, and 120 h of incubation, respectively. indicate that in SCC12 cells, which are malignantly transformed keratinocytes, the accelerated We found that SCC13 cells exhibit RolDH, RalDH, and SDH activity while lacking the cleavage of atRA could be one cause for insufficient retinoid receptor functioning and thereby expression and inducibility of the CYP26. After ROL-stimulation, RAL and RA-isomers appeared malignant proliferation characteristics of this cell line, whereas in SCC13 cells other factors appear while 4-oxo-RA-metabolites were not traceable. After RAL-induction, SCC13 cells formed high to be responsible for the malignant features of this cell line. levels of ROL and low levels of RA-isomers, but no. 4-oxo-RA metabolites, too. Even after stimulation with either RA-isomer, no CYP26 activity was detectable neither on the biochemichal (RP-HPLC) nor on the molecular level (rt-PCR). We conclude that SCC13 cells are able to interconvert ROL and RAL exhibiting inducible RolDH, RalDH, and SDH activity. Although they are able to generate RA-isomers from ROL and RAL (RalDH and CYP2 J4), they do not exhibit basal or inducible expression of the CYP26, which has been shown to 4-hydroxylate all- trans RA to 4-oxo-all-trans-RA in other cell lines (SCC12, HaCaT, MCF7, HL60). Since normal human epithelial keratinocytes also lack the expression of CYP26, this suggests that – in contrast to other malignant tissues – the pathways of retinoid metabolism and the activity and inducibility of the enzymes involved here do not seem to be severely altered in the SCC13 cell line.

285 286 Methylation Analysis of Patched Gene Promoter in Basal Cell Carcinoma. Treatment of Murine Skin Squamous Cell Carcinoma with Interferon Coincides with p53 Normal- Osamu Tago, Hiroshi Fujiwara and Masaaki Ito ization Department of Dermatology, Niigata University School of Medicine, Niigata, Japan L.-S. Loo, S. B. Tucker and Y. Tong Patched gene mutation is related with basal cell nevus syndrome; it has been reported that less Department of Dermatology, University of Texas Medical School, Houston, Texas than 30% sporadic basal cell carcinoma (BCC) cases are also associated with the gene mutation, Recent studies have shown that intradermal injection of interferon alpha (IFN-01α) is highly however, the etiology of the majority of sporadic BCC cases are yet to be elucidated. Genome effective in the treatment of human squamous cell carcinoma (SCC) and basal cell carcinoma methylation is one of the epigenetic regulation mechanisms of gene expression, and may control (BCC). However, there have been no reports about the effects of IFN-01α in a murine skin patched gene expression and development of BCC. tumor model. We analyzed promoter region of patched gene in four cases of BCC. DNA was extracted from Five–6-wk-old female Sencar mice, whose dorsal hair was shaved prior to initial exposure and BCC and from the adjacent normal skin separated under the microscope. The extracted DNA every 2 wk, were irradiated with UVA and UVB three times per week for 25 min per exposure was treated with or without HpaII, methylation-sensitive restriction enzyme, and patched gene (7.0 01ϫ 105 J per m2 per wk using the Dermalight 2001 Sun Lamp). When skin tumors promoter region was ampliﬁed by means of polymerase chain reaction. After electrophoresis PCR developed to 1.5–2.0 mm in size, UV treatment was stopped. m-IFN-01α and normal saline products were analyzed with densitometry using NIH Image software. The difference of tumor/ were injected intradermally around the tumor in two groups for 6 wk and another group had no normal skin was 7% in maximum. We could not ﬁnd the evidence that patched gene promoter treatment of tumors. Expression of p53 protein was evaluated by immunohistochemistry at the methylation is related with BCC development. site of skin tumor growths after 6 wk of tumor treatment. m-IFN-01α treatment caused considerable tumor regression. The nontreated group, the normal saline treated group, and the m-IFN-01α treated group had 0%, 0%, and 88% tumor regressions, respectively. The expression of p53 was seen in 100% of normal saline treated group and control tumors, but only 12% of m-IFN-01α treated group. (i) Intradermal injection of m-IFN-01α into mouse SCC causes tumor regression. (ii) Regression of SCC with intradermal m-IFN-a coincides with negative p53 staining.

287 288 Loss of Heterozygosity on the Long Arm of Chromosome 10 in Primary Cutaneous T- Antimetastatic and Antiproliferative Action of Resveratrol on Melanoma Cell Lymphoma J. Lynott, N. Chulamorkodt and R. Kumar J. Scarisbrick, A. Woolford, R. Russell-Jones and S. Whittaker University of Illinois at Chicago, College of Medicine, Department of Dermatology, Chicago, Skin Tumour Unit, St. John’s Institute Dermatology, London, UK Illinois Previous cytogenetic studies of primary cutaneous T-cell lymphoma (CTCL) have identified a Resveratrol, a phytoalexin found in grapes and other plants originally discovered in Peru in 1974, region on 10q23–26 that may be deleted. We have analysed CTCL tumour DNA samples was later identified as the active principle in Leguminosae that inhibited cyclooxygenase (COX) using a series of highly informative microsatellite markers on 10q23–26 to detect loss of activity. Like many other drugs that inhibit the activity of COX, resveratrol was previously shown heterozygosity (LOH). to inhibit tumor initiation and promotion in in vitro sysytems. In our experiments, resveratrol in LOH studies were performed on 54 paired tumour DNA and normal DNA samples from its pure form was examined in an in vitro culture system and in a murine melanoma model for its individual patients. The 54 tumour DNA samples consisted of Mycosis fungoides (n ϭ 44; clinical antimetastatic and antiproliferative effects In the murine model used, mice were inoculated with stage: T1 ϭ 13, T2 ϭ 16, T3 ϭ 13, T4 ϭ 2), Sezary syndrome (n ϭ 6) and other primary 800 000 B16 F1 melanoma cells intramuscularly on day zero and placed in one of three random cutaneous T-cell lymphomas (n ϭ 4). In total seven microsatellite markers on 10q23–26 were groups. The control group (A, N ϭ 4) received vehicle QOD while the delayed treatment group amplified by polymerase chain reaction with incorporation of p33 -dCTP prior to analysis by (B, N ϭ 6) received vehicle on days 1, 3, 5, and 7, and 1 mg of resveratrol on days 9, 11, 13, polyacrylamide gel electrophoresis. Autoradiographs were subjected to densitometry. LOH was and 15. The full treatment group (C, N ϭ 5) received 1 mg of resveratrol QOD. On day 17, recorded as a 50% or more reduction in one allele. All tumour DNA samples showing LOH were the animals were sacrificed and all the tumors were dissected out and weighed. Melanoma repeated for confirmation. metastasized to the peritoneal cavity in 100% (four of four) of group A. Only 55% (six of 11) of Eight of 44 (18%) of patients with MF had LOH on lOq: two of 29 (7%) with stage T1/2 and the mice treated with resveratrol had metastases to the peritoneal cavity. The average tumor size six of 15 (40%) with stage T3/4 disease showed LOH in this region. The minimal overlapping in group A was 3.19 (Ϯ 0.34) grams, in group B was 1.79 (Ϯ 0.83) grams, and in group C was region was 10q23.3–24. No LOH was detected on 10q in patients with Sezary syndrome or other 1.69 (Ϯ 1.02) grams. Resveratrol at a concentration of 100 µM over a period of 96 h inhibited forms of primary CTCL. the growth of melanoma in cultures by 63% (Ϯ 10%). These preliminary data warrant a These results indicate a relatively high frequency (18%) of LOH in MF which suggests that a more detailed investigation of resveratrol as a possible antimetastatic and antiproliferative agent putative tumour suppressor gene(s) on this chromosomal region may be important in the towards melanoma. pathogenesis of mycosis fungoides. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 571

289 290 Prostaglandin E2 Biosynthesis and Cyclooxygenase 2 Expression are Up-Regulated in Human Existence of Y-Chromosome-Positive Cells in Skin Biopsy of Female Systemic Sclerosis Patients Epidermal Cancers H. Fujiwara, O. Tago and M. Ito Y. Higashi, T. Kanekura and T. Kanzaki Department of Dermatology, Niigata University School of Medicine Niigata, Japan Department of Dermatology, Kagoshima University Faculty of Medicine, Kagoshima, Japan Etiology of systemic sclerosis (PSS) is unknown; because chronic graft-versus-host disease may Recent studies have validated the role of arachidonic acid cascade in cancer development in reveal similar sclerotic skin change, it is speculated that fetal blood cells could transfer into the several tissues. In the present study, we investigated the prostaglandin E2 (PGE2) production in maternal body during the pregnancy, may remain in the body, and provoke GVH-like reaction human epidermal cancer cells with examining the protein expression of cyclooxygenase-1 (COX- and sclerotic change of the skin in female PSS patients. 1), cyclooxygenase-2 (COX-2), and cytosolic phospholipase A2 (cPLA2); the rate-limiting enzymes Skin biopsy of female PSS patients were subjected to detection of Y-chromosome DNA by mean regulating PGE2 biosynthesis. We used human cutaneous squamous cell carcinoma cell line, HSC- of polymerase chain reaction followed by Southern blot, and of in situ hybridization analysis. Four 5, eccrine carcinoma cell line, EcCa, and a nontumorigenic human keratinocyte cell line, HaCaT, out of nine skin biopsies were positive for Y-chromosome DNA. Peripheral blood mononuclear as a benign counterpart. cells were also analyzed in ﬁve patients, and one case was positive. All of the positive subjects More than 50-fold PGE2 was produced by HSC-5 and EcCa when compared to HaCaT. While have at least one male offspring. In control seven of 32 of female normal skin showed positive cPLA2 and COX-1 protein were constitutively expressed in HaCaT, expression level showed no for Y-chromosome. The residual fetal cells transferred during pregnancy may be associated with increase in HSC-5 and EcCa. In contrast to cPLA2 and COX-1, COX-2 protein expression was the development of PSS. up-regulated by more than 4-fold in HSC-5 and EcCa in comparison with HaCaT. These results suggest that COX-2 plays a major role in mediating human epidermal cancer devel- poment.

291 292 Role of TGF-01β2 in Anagen-Catagen Transition of Human Hair Cycle The Retention of Human Sebaceous Gland Differentiation on Organ Culture for 7 d: the Effects T. Soma and T. Hibino of IL-101α and TGF01β2 Shiseido Research Center, Yokohama, Japan M. Downie and T. Kealey It has been suggested that several cytokines and growth factors were involved in human hair cycle. Department of Clinical Biochemistry, University of Cambridge, Cambridge, UK Among them, TGF-01β is suggested to play important roles in anagen-catagen transition. Indeed, Human sebaceous glands can be organ maintained over 7 d with no measurable loss of lipogenic our organ culture study clearly demonstrated that TGF-01β could induce catagen-like morphology. function (Guy et al, 1996, J Invest Dematol 106:454–460). To determine if organ culture will retain Here, we analyzed changes of TGF-bs and their receptors in human hair cycle. In addition, effect immunohistochemical markers of differentiation, glands were maintained in supplemented Williams of TGF-01β antagonist on the growth and morphology of hair follicles was investigated. Human E medium for 7 d. scalp specimens were obtained from plastic surgery. Formalin-fixed and paraffin-embedded tissue The expression of epithelial membrane antigen, a sebocyte-specific marker, was retained on organ sections were used in all immunohistochemical staining. In anagen hair follicles, TGF-01β2 culture. Freshly isolated sebaceous glands demonstrate very little expression of the keratinocyte immunoreactivity (IR) was restricted in the outermost layer cells of the outer root sheath (ORS). markers profilaggrin, profilaggrin/filaggrin, loricrin and involucrin, but these are upregulated on TGF-01β3 IR was observed in the keratogenous zone in anagen hair follicles. Strong TGF-01β2 7 d organ maintenance (n ϭ 5 subjects, five glands per subject). This suggests that sebocyte IR was detected in the lower bulb matrix cells adjacent to the dermal papilla (DP) in anagen- differentiation is altering towards a keratinocyte phenotype on culture. catagen transition phase, and then this staining disappeared as progression of catagen. In late When human sebaceous glands were maintained for 7 d in 0.01–10 ng per ml IL-101α, their catagen, regressing epithelial strands were positively stained by anti-TGF-01β2 antibody and anti- rates of lipogenesis were highly significantly reduced, although their rates of DNA synthesis were TGF-01β type II receptor antibody. These staining patterns were correlated well with TUNEL not affected (n ϭ 5 subjects, five glands per subject, respectively). IL-101α altered sebaceous gland staining in regressing hair follicles. Specific staining for anti-TGF-01β3 antibody seen in anagen morphology, inducing a frank keratinocyte-like morphology. This was associated with a marked hair follicles was no longer observed in catagen hair follicles. When anagen hair follicles were upregulation of the immunocytochemical intensity of staining of involucrin (n ϭ 5 subjects, five cultured in the presence of TGF-01β2, a catagen-like morphological change was markedly glands per subject). The pattern of Ki-67 staining appeared normal (n ϭ 3 subjects, five glands accelerated compared to that in the absence of TGF-01β2. Characteristic cell death seen in catagen per subject). These results suggest that IL-101α accelerates the alteration of sebocyte differentiation was confirmed in the bulb matrix cells around the DP and the ORS cells in hair follicles into a keratinocyte-like phenotype. This may explain many features of the sebaceous injury response. treated with TGF-01β2. Anti-TGF-01β neutralizing antibody partially prevented a catagen-like 0.1 and 10 ng per ml TGF01β2 significantly inhibited sebaceous lipogenesis and rates of DNA morphological change of hair follicles culture in vitro. Hair follicle growth in vitro was markedly synthesis, but the glands retained their glandular morphology and Ki-67 staining pattern. TGF01β2 and significantly stimulated in the presence of fetuin, potent antagonist of TGF-01β. TGF-01β2 thus inhibits lipogenesis without promoting a keratinocyte-like phenotype. may be a key molecule in the anagen-catagen transition and the apoptotic cell death in catagen phase.

293 294 Human Dermal Fibroblasts from Chronically Photodamaged Skin Exhibit a more Differentiated/ Induction of Keratin 9 in Non-Palmoplantar Keratinocytes by Palmoplantar Fibroblasts Through Post-Mitotic Phenotype in Culture Mesenchymal–Epithelial Interactions S. Alaluf, H. Muir-Howie and M. R. Green Y. Yamaguchi, S. Itami, M. Tarutani, K. Hosokawa and K. Yoshikawa Unilever Research, Colworth Laboratory, Sharnbrook, Bedford, UK Department of Dermatology, Osaka University, Osaka, Japan We have previously shown that when singly seeded under atmospheric (20%) oxygen tension, a Palms and soles differ from other body sites in terms of clinical and histological appearance, significant proportion of human neonatal foreskin fibroblasts are induced to differentiate along a response to mechanical stress, and the distribution of keratin 9. Because keratin 9 is exclusively terminal cell lineage, moving from a mitotic towards a postmitotic phenotype. In contrast, when expressed in the palmoplantar suprabasal keratinocyte layers, it is considered a differentiation seeded under more physiological (4%) oxygen conditions this differentiation is largely prevented. marker of palms and soles. We previously reported the palmoplantar mesenchymal influences on Consistent with these findings, it was also shown that depletion of the intracellular antioxidant keratin 9 mRNA induction in nonpalmoplantar keratinocytes. In the present study, we further glutathione strongly promotes terminal differentiation, indicating that this process is accelerated investigated this regulation both in vitro and in vivo. While nonpalmoplantar keratinocytes did not by oxidative stress. The aim of the current study was to determine whether dermal fibroblasts express keratin 9 mRNA, they expressed it when cocultured with palmoplantar fibroblasts by from chronically photodamaged skin exhibit an altered pattern of differentiation when compared northern blotting. When these cocultures were grafted on the dorsal muscle fascia of SCID mice, with fibroblasts from photoprotected skin. Human dermal fibroblasts were explanted from paired the epidermis showed histologically hyperkeratosis and acanthosis and immunohistochemically biopsies taken from high/moderate to severely photodamaged dorsal forearm and photoprotected expressed keratin 9. When pure epidermal sheets were grafted on palmoplantar skin defects due upper inner arm skin, respectively. Early passage cells were seeded into 96 well plates at a density to burn, injury, and acral lentiginous melanoma, the epidermis gradually demonstrated adoption of 0.8 cells per well and incubated for 2 wk in DMEM/10% foetal calf serum and 5% CO2 in a of palmoplantar phenotype and expressed keratin 9. Pure epidermal grafting may be effective in the 4% (physiological) O2 incubator. Cells were then stained with amido black and subsequent treatment of palmoplantar skin defects as a result of heterotypic mesenchymal–epithelial interactions. microscopic analysis revealed the presence of three morphologically distinct cell types: Type 1,a highly proliferative cell type in excess of 100 cells/well; Type 2, a transitional culture moving from a mitotic to a postmitotic cell type, with typically 10–100 cells per well; Type 3, a postmitotic cell with fewer than 10 cells per well. Comparison of these fibroblast populations revealed a statistically significant shift towards a more highly differentiated/postmitotic phenotype in cells from photodamaged versus photoprotected skin: Type 1 78.1 Ϯ 19.5.0% (protected) vs 66.2 Ϯ 18.3% (damaged, p ϭ 0.006, n ϭ 3); Type 2 12.0 Ϯ 12.1% (protected) vs 16.9 Ϯ 12.6% (damaged, p ϭ 0.09, n ϭ 3); Type 3 9.9 Ϯ 7.4% (protected) vs 17.0 Ϯ 6.4% (damaged, p ϭ 0.03, n ϭ 3). These results suggest that fibroblasts from chronically photodamaged skin exhibit a more terminally differentiated/postmitotic phenotype, consistent with previous exposure to elevated levels of oxidative stress. This phenotypic change may play a role in mediating the altered properties of the dermal extracellular matrix in chronically photoaged skin. 572 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

295 296 The Expression of Retinoic Acid Receptor 01α, Retinoid X Receptor 01α and Thyroid Hormone Regulation of Cell Death in Human Hair Follicle Cells by Caspases Receptor 01α is Reduced in Psoriatic Lesions as Compared to Nonlesional Skin M. E. Sawaya,*† U. Blume-Peytavi,† D. L. Mullins,‡ D. W. Nicholson§ and R. W. Keane*ϩ H. To¨rma¨, T. Karlsson, G. Michae¨lsson, O. Rollman and A. Vahlquist *Department of Biochemistry & Molecular Biology, University of Miami, Florida; †Department Department of Medical Sciences, Section of Dermatology, Uppsala University, Sweden of Dermatology, Freie Universitat Berlin, Germany; ‡Department of Pathology, Memorial Mission Retinoic acid, vitamin D3 and triiodothyronine are all involved in the regulation of keratinocyte Hospital Asheville, North Carolina; §Research Division Merck Frosst Co, Quebec, Canada, proliferation and differentiation, i.e. processes that are disturbed in lesional psoriatic skin. The ϩDepartment of Physiology & Biophysics effects are mediated via nuclear hormone receptors; viz. the retinoic acid receptors (RAR-01α, Recent studies have revealed that apoptosis is involved in the dynamic cycles of hair growth. γ α β Ϫ01 ), the vitamin D3 receptor (VDR), the thyroid hormone receptors (TR-01 , Ϫ01 ) and Caspases are intracellular proteases that function as initiators and effectors of apoptosis. Using a the common heterodimer partners, the 9-cis retinoic acid receptors RXR-01α and Ϫ01β.By panel of anticaspase antibodies, caspase-3 was localized immunchistochemically in human scalp using a quantitative real-time PCR the mRNA expression of these receptors and three different skin to the basal epidermis, outer sheath of hair follicles, matrix and dermal papilla, eccrine and house-keeping genes (cyclophilin, GAPDH and 01β-actin) was studied in psoriatic skin. Since sebaceous glands. These structures did not stain for anticaspase-1 or Ϫ2 antibodies. Purified the expression of the house-keeping genes was constantly 2.7–4.4 times higher in lesional as populations of dermal papilla, dermal fibroblasts, keratinocytes, and melanocytes obtained from compared to nonlesional skin one of them (01β-actin) was used to normalize the expression of human adult skin were induced to undergo apoptosis by treatment with 0.5 µM staurosporin and the nuclear receptor genes. Thus, the mRNA levels of RAR01α, RXR01α and TR01α in confirmed caspase-3 activation in these cell types. Time-course experiments show that activation lesional skin were found to be decreased by 58, 70, and 75%, respectively, while RAR01γ, VDR, of caspase-3 in most skin cell types occurs within 1 h following staurosporin treatment with the and TR01β were not significantly altered. As a consequence, the ratio of RXR01α:RAR01γ, exception of keratinocytes which demonstrated cleavage of procaspase-3 after 16 h of staurosporin which was 3.3 in nonlesional skin, decreased to 1.3 in lesional skin. The results suggest that treatment. In addition, cleavage of caspase-2 was detected in extracts of dermal papilla and dermal retinoid and thyroid hormone signaling systems are altered in lesional psoriatic skin. fibroblasts after 1 h of staurosporin treatment. These studies demonstrate that caspases can directly regulate apoptosis of hair follicle cells which may play an important role in signaling various stages of the hair cycle affecting hair loss.

297 298 The New Molecule Phytosterol Sulfate is a Membrane Stabilizer and a Moderator of P53 Disappearance of Actin Stress Fibres Occurs Before Nuclear Condensation in Oxidative Stress- Expression after UV Exposure Induced Cell Death of Dermal Fibroblasts N. Domloge, S. Marguerie, A. Geffard, K. Golz and L. Zastrow E. Ulukaya and E. J. Wood Department of Dermatology and Skin Care Research, Coty International Research and Develop- University of Leeds, School of Biochemistry and Molecular Biology, Leeds, UK ı and H ment Center, Monaco Cells can be injured by oxidants such as O2 –ı 2O2 which are produced as a result of Keratinocyte differentiation and stabilization of cell membrane lipid bilayer, are two important inflammation in vivo. However, pathway(s) of oxidant stress are not well understood yet. The conditions for cell protection from external stress. Phytosterol sulfate (PS), a new homologue to effects of exogenous H2O2 on human dermal fibroblasts in monolayer culture on either collagen- Cholesterol sulfate (CS) which plays a role in cell membrane protection, has been developed and coated or uncoated coverslips were studied. Cytotoxicity was assessed by the MTT assay, the rate shown to induce cell differentiation in a similar manner to CS. The effect of PS on human cells of DNA synthesis was measured by [3H] thymidine incorporation, morphological changes occuring under membrane destabilizing conditions and the protection capacity of PS-differentiated cells upon cell death were visualised by hematoxylin staining, F-actin was stained with FITC-labelled against UV, were investigated. Studies on cultured human cells and skin organ cultures were phalloidin and integrins were detected immunocytochemically. H2O2 inhibited DNA synthesis examined by different staining methods, immunoblotting and image processing. Cultured human and induced cell detachment, cell shrinkage and nuclear condensation which are features of keratinocytes and fibroblasts were pretreated with PS followed by 10 µg per ml of SDS for 24 h. apoptosis-like cell death, in a dose-(45–780 01µM) and time-dependent manner. 01β1-Integrin The results showed membrane protection effect in PS-treated cells with few SDS morphological was expressed more prominently than other types (01β4, 01α2, 01α3, 01α5, 01αv01β5) and its changes and only 10% loss of cells; by comparison, in untreated control cells, SDS morphological distribution was changed by H2O2 treatment, resulting in small local foci of staining intensity. µ changes were predominant and 60% of cell were lost. P53 Immunostaining and immunoblotting Treatment with 780 01 MH2O2for 3 h caused actin stress fibres to be disrupted and stain less studies on human keratinocytes and fibroblasts exposed to a single UVB dose of 100 mJ per cm2, intensely, which may be due to cleavage by proteases, while there was no change in the size of using antibodies to mutant and wild type P53, demonstrated a moderate increased expression of nuclei. After 5 h cells retracted and their actin fibres had almost totally disappeared, although P53 (40%) in cells pretreated with PS 12–96 h prior to UV irradiation, compared to the 80% there were some remaining connections of actin at the borders of cells: also nuclear condensation increase in the control PS-untreated irradiated cells. Studies at different time points using light could only be observed after this stage. This indicates that cytoskeletal disruption is preceded by microscopy, differentiation markers and phospholipid staining methods showed that the majority nuclear condensation in fibroblasts subjected to oxidative stress. This might mean that the of PS-treated cells preserved their differentiated morphology and viability, and resisted UV damage disruption of actin plays a role in the activation of nucleases. compared to the PS-untreated irradiated control. This protective effect was confirmed by different immunostaining studies on skin organ cultures exposed to UV, after the application of PS or placebo formulae. These results demonstrate the interesting role of the new molecule Phytosterol Sulfate in protecting the cells, from harmful conditions such as SDS and UV stress, probably by enhancing their differentiation, membrane stability and phospholipid content.

299 300 Oxidative Stress Response in Normal Human Skin Versus Noninvolved Atopic Dermatitic Skin Binding of cAMP to RI Subunit of Erythrocytic cAMP-Dependent Protein Kinase A in Active C. Mundt, T. Blatt, R. Wolber, G. Gercken,† H. Fo¨lster-Holst,‡ D. Schachtschabel* and F. Sta¨b Psoriasis but Not in other Inflammatory Dermatoses is Decreased PGU Skin Research Center, Beiersdorf AG, Hamburg, Germany; *Clinical Department of R. E. Schopf, Y. Langendorf, R. E. Benz, P. Benes Physiological Chemistry, University Marburg, Germany; †Department of Biochemistry, University Department of Dermatology, Johannes Gutenberg University, Mainz, Germany Hamburg, Germany; ‡Clinical Department of Dermatology, University Kiel, Kiel, Germany To test whether a diminished binding of cAMP to protein kinase A (PKA) in erythrocyte 3 Free radicals are supposed to play a significant role in inflammatory skin diseases. In previous membranes is specific for psoriasis, we compared the binding of 2- H-8-N3-cAMP (cAMP) to in vivo investigations on the antioxidant state of human skin using the UVA-induced ultra-weak the RI subunit of PKA in erythrocyte membranes in five groups of individuals: 24 normal controls, photon emission technique (UPE) we found a significantly reduced antioxidant state in noninvolved 14 patients with atopic dermatitis, 15 with other, nonatopic inflammatory skin diseases (eczema, atopic dermatitic skin compared with normal healthy skin. The present ex vivo/in vitro data erythroderma, tinea, Grover’s disease, erysipelas, urticaria), eight other dermatoses mediated by demonstrate some specific differences on the cellular level in the oxidative stress response (UVA, immune mechanisms (lupus erythematosus, disseminated lichen planus, necrotizing vasculitis, H2O2) of normal versus atopic skin. In these studies we investigated the keratinocyte and the erythema nodosum, systemic sclerosis), and 31 with psoriasis as assessed by the psoriasis area and fibroblast fractions isolated from skin biopsies of panelists either with normal healthy skin (n ϭ severity index (PASI-score). Binding of cAMP to the regulatory subunit RI of PKA in erythrocyte 16) or noninvolved atopic dermatitic skin (n ϭ 7) by measuring the intracellular levels of phospho- membranes was measured by determining the UV-light catalized incorporation of cAMP into tyrosines (PT), thiols (THS) and the mitochondrial membrane potential (MMP) as well as the PKA evaluated by Scatchard plots. We found the following femtomolar binding per mg protein: enzymatic activity of intracellular PT-kinases (PTK) and PT-phosphatases (PTP) of primary human controls, 1025 Ϯ 22 (mean Ϯ SE); atopic dermatitis, 960 Ϯ 28 (n.s., two-tailed Wilcoxon test fibroblasts and keratinocytes, respectively. Our data indicate that in general primary keratinocytes compared to controls); nonatopic inflammatory diseases, 980 Ϯ 25 (n.s.); immune dermatoses, are significantly more resistent against oxidative stress (UVA, H2O2) than primary fibroblasts and 1005 Ϯ 36 (n.s.); psoriasis 645 Ϯ 31 (p Ͻ 0.0001). Treatment of psoriasis either topically with the constitutively expressed PTK-and PTP-activities are significantly enhanced in atopic dermatitic anthralin or systemically with etretinate or cyclosporin A normalized the binding of cAMP which skin cells. Furtheron we found in the fibroblast fractions derived from noninvolved atopic skin a was inversely correlated to the PASI-score. We conclude that the highly deceased binding of significantly higher stress responsiveness concerning the parameter THS, MMP, PTK and PTP, cAMP to MA in psoriatic erythrocyte membranes is disease-specific and normalizes after different whereas in the keratinocyte fractions no significant differences in the oxidative stress response forms of successful treatment. could be observed between keratinocytes from normal and noninvolved atopic skin. However, a pretreatment of skin cells for with antioxidants (i.e. alpha glucosylrutin) resulted in a significant protection against oxidative stress induced modulation of the parameters investigated. These data indicate that concerning oxidative stress responsiveness the primary functional differences between normal and atopic skin seem to be predominantly localised in the dermal layer and can be modulated by antioxidants. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 573

301 302 Catabolic Enzymes Involved in Stratum Corneum Barrier Formation: Activities in Human Heterogeneity in Clonal Potential and Life Histories of Individual Keratinocytes Propagated in Epidermal Keratinocytes and Porcine Epidermis Serum-Free Medium A. Schepky, R. Siegner, U. Holtzmann, R. Escola, W. Diembeck, R. Wepf,* U. Hintze* and J. J. Wille G. Vielhaber* Hy-Gene, Inc, Trenton, New Jersey Department of Biocompatibility/Biochemistry and *Department of Central Analytical, Beiersdorf The clonal life histories of over 450 individual clones were studied. Individual basal keratinocytes AG, Hamburg, Germany were derived from dividing cultures, previously cultivated in low calcium (0.1 mM) complete The generation of the epidermal permeability barrier is accomplished by a range of enzymes as serum-free medium, by seeding 1000 cells into 100 mm Petri dish containing cloning chips and 01β-hexosaminidases (A ϩ B), 01β-glucosylceramidase, acid phosphatase, lipases/esterases and prewarmed sterile complete serum-free medium. Chips with a single attached cell were identified stratum corneum chymotryptic enzyme. We are interested in the characterization of these enzymes and transferred to individual 30 cm2 Petri dishes using an inverted phase microscope placed in a found in human epidermis. We first established enzyme assays employing optimized human sterile cabinet. The dishes were refed pre-warmed low calcium or high calcium (2 mM) complete epidermal keratinocyte cultures (HEK) due to only very few useful data available in the scientific serum-free medium. All dishes were monitored several times a day and the number of cells in literature. For further characterization, porcine epidermis was used due to limited availability of each colony tallied daily for further analysis. A total of 369 clones were established from seven human epidermis. This study clearly demonstrates that all enzymes tested here in ammonium- human neonatal foreskin cell strains (A-G) and 113 clones were derived from one human adult chloride-extracts of excised porcine epidermis are still active after this new developed preparation skin cell strain (H). During a five day period, among the 32 clones of strain A, 83% divided at procedure. Moreover, the activities of the investigated enzymes in human keratinocyte cultures least once; 50% divided once in 24 h, 86% divided at least three times within 3 d, and more 50% and in porcine epidermis are comparable. Further experiments are underway to correlate these divided at least four to five times in 5 d. Of the 231 clones distributed amongst strains B–F, an data with those obtained with human epidermis. Results of these studies should give additional average of 63% (Ϯ 12 SD) had a high proliferative potential, i.e. divided three or more times guidance for the design of appropriate cosmetic products intended to trigger defined skin over an 8-d period. The less proliferative clones either divided only once, twice or not at all. Of enzymes’ activities. the 106 clones of cell strain G reared in high calcium medium 67% (71 of 106) divided more than three time in a 6-d period, and 55% (58 of 106) divided five or more times in 6 d. Clones derived from adult skin had a lower clonogenic potential, only 48% divided at least once in 7 d, and only 16% (18 of 113) had divided three or more times. This may reflect the relatively slower average doubling time (GT 01ജ 48 h) of keratinocytes derived from this third passage adult skin culture. The GT for second and third passage neonatal skin cultures was 24 h. Detailed dendrograms were constructed for many proliferating clones. Most clones displayed a holochronic division pattern, i.e. every cell dividing once a day. In several clones every cell divided more than once a day (e.g. GT ϭ 21.6 h), Many clones displayed multiphasic division patterns with every cell dividing once a day, then abruptly slowing to once every other day. Conversely, some clones failed to divide at first, then divided twice the next day, and then reverted to dividing once per day for the next several days. These results suggest dynamic behavior typical of limit cycle oscillator control of division timing.

303 304 Antioxidant Regulation of IFN-01γ-Induced ICAM-1 Expression in Human Keratinocytes Perturbed Assembly of the Cornified Cell Envelope in Cultured Keratinocytes and Papilloma T. Bito, S. Roy, C. K. Sen and L. Packer M. Jarnik, U. Lichti, M. Simon and A. C. Steven Department of Molecular and Cell Biology, University of California at Berkeley, California LSB, NIAMS, NIH and LCCTP, NCI, NIH, Bethesda, Maryland; and Department of Biology, Cutaneous inflammatory reactions are associated with the recruitment of circulating leukocytes Brookhaven National, Laboratory, Upton, New York, New York into the tissue. Such recruitment depends on the expression of cell adhesion molecules (CAM) in The cornified cell envelopes (CE) of different squamous epithelia of the mouse studied to date keratinocytes. Human epidermal keratinocytes have been observed to markedly express ICAM-1 are very similar with respect to surface texture, physical thickness, and mass per unit area but during inflammatory skin diseases. Intracellular redox state have been shown to modulate expression differ somewhat in protein composition depending on body site. To further investigate variability of CAM. We investigated the effect of various antioxidants (thiol, phenolic and flavonoid) on in CE assembly we have studied CEs purified from cultured murine keratinocytes induced to IFN-01γ-induced ICAM-1 expression in primary adult human keratinocytes (HK cells) and differentiate by calcium and CEs from chemically induced papillomas. Techniques used were (i) HaCaT cells using flow cytometry. IFN-01γ (20 U/mL) induced twofold ICAM-1 expression in protein composition modeling based on amino acid composition, and (ii) dark-field scanning HK cells and threefold expression in HaCaT cells. TNF-01α (20 ng per ml), potent inducer of transmission electron microscopy for measurement of mass-per-unit-area. The CEs of cultured ICAM-1 in endothelial cells, did not affect ICAM-1 levels in keratinocytes. Pre-treatment of keratinocytes are, on average, only half as dense as native CEs (3.5 vs 7.0 kDa per nm2) and are keratinocytes with 01α-lipoate (50 or 100 µM), 01α-tocopherol (50 µM), taxifolin (50 or 100 markedly less regular. Their amino acid composition is much lower in Gly/Ser, suggesting a µM) or a French maritime pine bark extract (PBE), Pycnogenol (20 or 50 µg per ml) downregulated reduced content of loricrin and an enhanced content of other CE proteins. Papilloma CEs are IFN-01γ-induced ICAM-1 expression. The ICAM-1 promoter region has several NF-01κB and also less dense than native CEs (~6 kDa per nm2), and their amino acid compositions also imply IRF-1 sites. 01α-Lipoate or 01α-tocopherol did not influence the activation of IFN-01γ-mediated a reduced loricrin content. Preliminary observations on immature native CEs suggest that CE transcription factors NF-01κB and IRF-1, however, downregulation of IFN-01γ-mediated assembly is normally processive and regulated so as to produce a uniform, unbroken layer of cross- activation of NF-01κB and IRF-1 was observed by taxifolin or PBE treated cells. The inhibitory linked protein. When this process is perturbed, the CEs produced are thinner, less regular, and effect of antioxidants on IFN-01γ-induced expression of ICAM-1 could have potential therapeutic lesion-prone. We suggest that these differences arise from altered supplies of loricrin and other value in skin disorders. CE precursor proteins and/or altered activity of the transglutaminase enzymes that perform the cross-linking.

305 306 Water Activity: The Critical Factor Controlling Stratum Corneum Chymotryptic Enzyme (SCCE) The Cornified Cell Envelope: An Important Marker of Stratum Corneum Maturation in Healthy And Desquamation and Dry Skin A. Watkinson, J. Rogers and C. Harding C. Harding, S. Long, J. Rogers, J. Banks, Z. Zhang* and A. Bush* Cell Biology & Physiology Unit, Unilever Research, Colworth Hse, Bedford, UK Cell Biology & Physiology Unit, Unilever Research, Colworth House, Bedford, UK; *Department Stratum corneum chymotryptic enzyme (SCCE) is a differentially regulated serine protease which of Chemical Engineering, University of Birmingham, Birmingham UK is localized within the lipid-rich intercellular spaces (ICS) of the stratum corneum (SC) suggesting The cornified cell envelope (CE) formed by transglutaminase mediated 01ε-(01γ-glutamyl) a role in desmosomal degradation to facilitate desquamation. Since the ICS is a water-poor lysine cross-linking of specialised proteins is the most insoluble component of the terminally environment we have investigated the relationship between water activity (WA), SCCE activity differentiated keratinocyte. and desquamation under defined conditions of relative humidity (RH). Human skin, embedded Under Nomarsky optics two types of CE are readily distinguishable: an irregularly shaped, readily in agar and exposed to defined RHs, was incubated for 48 h, after which biopsies were taken and deformed ‘‘fragile’’ envelope (CEf) which predominates in the deeper layers of the stratum the functionally desquamated surface corneocytes recovered and quantified. In this model system corneum, and a polygonal ‘‘resilient’’ or ‘‘rigid’’ envelope (CEr), which represent over 80% of the desquamation was maximal at 96% RH but was markedly inhibited at 33% RH and inhibited by CE population in the superficial layers. This distinct spatial distribution indicates a maturation of Zn2ϩ, a potent inhibitor of SCCE. To determine whether the desquamatory rate in vitro reflected the CE from the fragile to the resilient phenotype during stratum corneum maturation. In this the relative activity of SCCE, we investigated the activity of this protease under similar condition study we have examined morphological and physical changes occurring in the CE during using the SCCE selective fluorogenic substrate suc-leu-leu-val-tyr-AMC. The substrate was terminal differentiation. topically applied to skin and incubated at defined RH at 37°C for 12 h, the surface stratum The proportions of CEf and CEr present in superficial samples of stratum corneum were readily corneum was recovered by tape-stripping and hydrolysis determined. At 17% RH, enzyme activity distinguishable following staining with Tetrarhodamine isothiocyanate (TRITC) and showed was virtually absent and markedly inhibited at 44% RH, compared with 96% RH. To investigate significant body-site variation. The percentage of CEf was highest on samples recovered from the relative influence of water activity on SCCE activity verses other serine-class proteases two exposed body-sites (back of hand Ͼ cheek Ͼ inner bicep) indicating that photodamage and other further assays were performed. Firstly, human recombinant SCCE (rSCCE) was incubated with environmental trauma reduce or delay normal CE maturation. Soap-induced dryness resulted in suc-leu-leu-val-tyr-AMC in a range of aqueous sucrose solutions equivalent to WA of 0.78–1.0. a significant decrease in CE maturation coincidental with reduced desmosomal hydrolysis. Effective Secondly, rSCCE activity was determined by casein zymography under a similar WA range. In moisturisation of dry skin enhanced CE maturation (33% increase in TRITC fluorescence. n ϭ both assays rSCCE was relatively resistant to the reduction of water activity. A decrease in WA 14 following 2 wk treatment). Using a novel micromanipulation instrument the force required to 0.9 lead to a 30% and 60% decrease in trypsin and chymotrypsin activity, respectively, whereas (01µN) to maximally deform individual CEf and CEr was compared. CEf recovered from deep SCCE activity remained unchanged. stratum corneum were significantly weaker than CEr recovered from superficial layers (62 Ϯ 7 In conclusion: (i) we have developed an in vitro model to determine the influence of RH and uN vs 900 Ϯ 140 uN, respectively). water activity on desquamation; (ii) shown that desquamation was RH dependant and inhibited These studies indicate that the normal process of CE maturation is associated with an actual by Zn2ϩ, a potent inhibitor of SCCE; (iii) shown that, compared to other serine proteases, rSCCE strengthening of this insoluble, protective structure and that the impairment of this process is is relatively refractile to decreases in WA, a property which may be essential to facilitate associated with poor quality of the stratum corneum. desquamation in the upper layers of the stratum corneum. 574 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

307 308 Pharmcological Regulation of Human Keratinocyte CD44 Expression and its Binding to Keratinocytes that Adhere Most Rapidly to Type IV Collagen and Display Characteristics of Stem Hyaluronic Acid Cells are Protected from Apoptosis M. Dumas, M. Huart and F. Bonte A. Marconi, R. Tiberio, C. Fumelli, A. Giannetti and C. Pincelli LVMH lab R&D, Branche Parfurns Cosmetiques, Saint Jean de Braye, France Department of Dermatology, University of Modena, Modena, Italy CD44, a transmembrane glycoprotein involved in extracellular matrix interactions, is the principal Normal human keratinocytes rapidly adhering to the extracellular matrix (ECM), expressing cell surface receptor that binds hyaluronan (HA). We have investigated pharmacological modulations highest levels of 01β1 integrin and with the greatest colony-forming efficiency (CFE) are thought using CD44 standard expression and hyaluronate binding by normal human keratinocytes in to be stem cells. In addition, disruption of cell–matrix interaction that induces apoptosis in certain culture treated with alpha hydroxy acids, retinoic acid and 1,25(OH)2 vit D3, molecules which cell types is mediated by integrins. The purpose of the present study was to investigate whether modulate skin differentiation and help treat skin aging. Human normal keratinocytes (10 cells per normal human keratinocytes, most attached to ECM, are protected from apoptosis. Primary well) were grown in HK-SFM (Gibco) medium in 96 multiwell plates (Costar) for 24 h. Then, keratinocyte cultures were divided into three populations: they were first allowed to adhere to new medium containing pharmacological agents was added for 48 h. The cell DNA content was type IV collagen for 5–20 min (population 1), and the nonadherent cells were then transferred to measured by the DAPI (401Ј601Јdiaminephenylindole-dihydrochloride) assay and cytotoxicity fresh collagen-coated dishes and allowed to attach overnight (population 2). Finally, keratinocytes with a neutral red viability assay. CD44 receptors, were detected by immunostaining in a PBS ϩ not yet attached after one night were plated onto type IV collagen in order to obtain a third 10% FCS, 0.05% sodium azide medium with fluorescent (phycoerythrin) anti-CD44 murine population (population 3). Adhesion to type IV collagen was blocked by preincubating keratinocytes monoclonal antibody, clone A3D8 (14 µg per ml, 30 µl per well). Competitive binding of HA with anti-01β1 integrin. CFE in population 1 was the highest after 14 d in culture, showing a 5- to its CD44 receptor was measured at 4°C with and a f luorescein – amine bound HA. D-L lactic fold increase over population 2, whereas CFE was virtually not detectable in population 3. acid at 2.2 01ϫ 10–4 M increased the CD44 expression by 25% and HA binding of 78%. Glycolic Population 1 expressed low levels of involucrin, while the number of involucrin-containing cells acid and gentisic acid increased also CD44 expression, i.e., ϩ25% for 4.1 01ϫ 10–5 M; ϩ45% progressively increased in population 2 and 3. 01β1 integrin was expressed in most cells of for 7.1 01ϫ 10–5 M, respectively. Citric acid had no effect and malic acid decreased standard population 1, while it decreased significantly in population 2 and 3. Percentage of apoptotic cells, CD44 expression (–25% for 1.4 01ϫ 10–4 M). At 10–9 M retinoic acid increased CD44 by 44% as measured by the TUNEL method at 48 h, was significantly higher in population 2 (8.6%) and and stimulated HA binding by 65% but decreased them at lower concentrations. 1,25(OH)2Vit 3 (29.2%) than in population 1 (1.6%), p Ͻ 0.001. A similar percentage of apoptotic cells was D3, at 10–7 M, which causes keratinocyte differentiation decreased CD44 by 40% but increased detected by flow cytometry. In population 3, apoptosis was also confirmed by the cleavage of HA binding by 26% at the same concentration. Our data establish that keratinocyte differenciation poly(ADP-ribose)polymerase. The addition of anti01β1 integrin, but not of antivimentin antibody agents can modulate CD44 expression and hyaluronic acid binding differently and independently. to both population 1 and 2 caused a significant increase in apoptotic cells. Bcl-2 protein levels were markedly higher in population 1 than in population 2, while in population 3 they were almost undetectable. After the addition of anti-01β1 integrin, % of apoptotic cells was significantly lower in HaCat cells overexpressing bcl-2 than in control transfectants. These results indicate that keratinocytes that adhere most rapidly to ECM and are considered to be stem cells are protected from apoptosis via a pathway that appears to be bcl-2 dependent.

309 310 Cathepsin D- and Chymotrypsin-Like Proteinases Participate in Desquamation at Acidic pH Thallium Inhibits Proliferation, Modulates Signal Transduction and Alters Terminal Differentiation S. Igarashi, T. Horikoshi, H. Uchiwa, H. Brysk* and M. M. Brysk*†‡ in Cultured Keratinocytes Basic Research Laboratory, Kanebo Ltd, Odawara, Japan; Departments of *Dermatology, †Micro- C. Krautmacher, D. Peus, K. Squillace, D. Schilling, J. Arbiser* and M. Pittelkow biology and Immunology, and ‡Human Biological Chemistry and Genetics, University of Texas Departments of Dermatology and Biochemistry and Molecular Biology, Mayo Clinic/Foundation, Medical Branch, Galveston, Texas Rochester, Minnesota; and *Department of Dermatology, Emory University School of Medicine, In vitro studies of desquamation have been traditionally performed at alkaline pH, even though Atlanta, Georgia the stratum corneum has an ambient acidic environment of about pH 5. The standard model Thallium (Tl) salts exert therapeutic as well as toxic effects on skin. We have previously shown system achieves squame shedding upon incubation of plantar stratum corneum for1dinan that Tl inhibits keratinocyte proliferation and alters terminal differentiation under high calcium alkaline buffer that must include a chelating agent. We demonstrate desquamation in vitro when conditions. We characterize the kinetics and reversibility of thallium-induced growth arrest in plantar callus is incubated for 4 d at pH 5, without addition of exogenous chelators. We show human keratinocytes, examine growth factor receptor and downstream signaling pathways as that in our system, desmoglein I becomes degraded. To determine which proteinases participate potential targets for Tl activity, and determine the roles of Tl and calcium in terminal differentiation. in squame shedding at acidic and basic pH, we added a variety of specific proteinase inhibitors. Tl causes potent, dose-dependent (25–1000 mM) growth arrest within 4 h as determined by [3H]- At pH 8, the crucial enzymatic activity was a chymotrypsin-like serine proteinase, while a similar thymidine incorporation. Clonal growth assay shows initial reversibility (within 9 h) of growth activity at pH 5 was accompanied by an aspartic proteinase activity of comparable strength. Using arrest and subsequent irreversible commitment. EGF receptor analysis shows late decrease (48–72 insulin B chain as a model protein, we incubated it with shed squames or commercial enzymes, h) in total and phosphorylated levels. However, ERK 1/2 activation is markedly inhibited at and added a variety of specific proteinase inhibitors. Four degradation peaks were observed when earlier times by Tl, whereas early activation of JNK is not observed. Tl causes only minimal, insulin B was reacted with shed squames at pH 5. Two of these peptides were suppressed by the delayed cell death. Northern analysis of keratin 1, keratin 10 and involucrin expression was addition of PMSF, the other two by pepstatin A; chymostatin inhibited all four, but E-64 and performed in confluent cultures. Under high calcium conditions, Tl promotes differentiation, leupeptin had no effect. The implied specificity was confirmed by reacting the insulin with human whereas under low calcium conditions, Tl inhibits expression of terminal differentiation markers. liver cathepsin D and pancreatic chymotrypsin, reproducing the expected degradation products. These findings demonstrate that Tl is a bio-active heavy metal that targets epithelium and induces Endogenous proteinases corresponding to these activities have been previously identified, namely early, reversible growth-arrest, coupled to inhibition of ERK1/2 activation. Irreversible loss of stratum corneum chymotryptic enzyme and the mature active form of cathepsin D. Our results proliferation is linked to terminal differentiation that is modulated by calcium concentration. Tl suggest that endogenous cathepsin D-like and chymotrypsin-like proteinases are involved in may be a probe to further delineate cellular control of epidermal homeostasis. epidermal desquamation at acidic pH.

311 312 Identification of a Novel Transmembrane-Protein (pKe#192/MAP17) Expressed by Human Activity of Thymidine Phosphorylase/Platelet-Derived Endothelial Cell Growth in Cultured Ker- Keratinocytes in the Upper Stratum Granulosum of the Epidermis atinocytes C. Jaeger P. M. Schwartz and J. G. Haggerty Department of Dermatology and Institute of Immunology and Serology, University of Heidelberg; Department of Dermatology, Yale University, New Haven, Conneticut; Dermatology, Service, BM Schaefer and MD Kramer, Institute of Immunology and Serology, University of Heidel- VA Conn. Health Care System, West Haven, Conneticut berg, Germany Thymidine phosphorylase/platelet-derived endothelial cell growth factor/(TPase/PD-ECGF) is With the aim to identify genes involved in activation of human keratinocytes, a subtractive cDNA an enzyme that is chemotactic for endothelial cells in vitro and is angiogenic in vivo. Immunohisto- library was generated from resting versus activated keratinocytes. Activation of human keratinocytes chemical data from skin samples indicated that TPase/PD-ECGF intensity increased with was caused by treatment with the enzyme dispase. One of the isolated genes was the clone differentiation. We have investigated the regulation of TPase/PD-ECGF activity by factors which pKe#192. Sequencing analysis of this cDNA clone revealed an open reading frame of 411 bp. By induce growth and differentiation in vitro. Small keratinocytes (0.6–0.9 pl), newly isolated from database analysis was ensured that this gene was never described before in human skin, however, human foreskin, have very low TPase/PD-ECGF activity (0.1–0.2 nmol thymine formed per 105 this gene was found in human kidney previously (termed ‘‘MAP17’’). Kyte-Doolittle hydrophobicity cells-30 min). The expression of TPase/PD-ECGF activity is sensitive to culture conditions. analyses showed a hydrophile amino terminus of 13 amino acids, a transmembrane region and a Keratinocytes, maintained in cMCDB with 0.1 mM Caϩϩ, range from 2–3 pl in volume with cytoplasmatic tail of 61 amino acids. The protein possesses two potential phosphorylation sites. TPase/PD-ECGF activity of 1–2 nmol thymine formed per 105 cells-30 min. Increasing the Regulation of pKe#192/MAP17 was proved with northern-blot analyses and rt-PCR using Caϩϩ concentration has very little effect on activity while including serum increases activity 3– mRNA of resting human keratinocytes and by dispase treatment activated keratinocytes. Northern 5-fold. When stratified keratinocytes, grown in DME with serum, are separated by size by blot analysis as well as rt-PCR showed weak expression in resting keratinocytes and increased elutriation, we can generate cell populations with an increasing degree of differentiation. Smaller, expression after stimulation. For further characterisation at the protein level pKe#192 was expressed less differentiated cells had low TPase/PD-ECGF activity whereas larger cells with increased in the GST-System. Pure recombinant pKe#192 was obtained by purification with Glutathion- involucrin expression showed progressively higher levels of TPase/PD-ECGF activity; over a 2– Sepharose columns and thrombin cleavage at the column. Recombinant pKe192 was used to 10 pl volume range activity increased from 4 to 6 nmole thymine formed to 35–40 nmole thymine generate mono-and polyclonal antibodies. The antibodies were tested on human kidney with formed per 105 cells-30 min. We also investigated how agents which induce differentiation effect western blot and immunohistology. Immunohistological studies using the mono-and polyclonal TPase activity. Methotrexate and TPA both increased TPase/PD-ECGF 3–10-fold on a per cell antibodies showed a specific staining of the upper layers of the stratum granulosum in normal basis. We conclude that TPase/PD-ECGF is regulated by keratinocyte culture conditions and human epidermis. The staining was colocalised with involucrin. In conclusion, these data suggest degree of differentiation. The regulation of TPase/PD-ECGF may be an important factor in a role of pKe#192/MAP17 in epidermal physiology. normal wound healing and in tumor angiogenisis. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 575

313 314 Forty Per Cent of Porcine Corneocyte Envelope 01ω-Hydroxyceramides are Bound to Protein Computer Modeling and Synthetic Peptides Indicate an 01α-Helical Conformation for Involucrin Through Their 01ω-Hydroxyl and Sixty Percent Through Their Sphingosine 1-Hydroxyl in the Corneocyte Envelope D. T. Downing, M. E. Stewart and N. Lazo N. Lazo and D. T. Downing Marshall Research Laboratories, Department of Dermatology, Univ of Iowa, Iowa City, Iowa Marshall Research Labs, Dept of Dermatology, University of Iowa, Iowa City, Iowa To fully define which of the hydroxyls of corneocyte envelope 01ω-hydroxy-ceramides are The mammalian epidermal corneocyte has a monolayer of 01ω-hydroxy-ceramide bound through involved in their binding to the protein envelope, we employed tri(isopropyl)silyl chloride (TIPS) ester linkages to the surface of the cross-linked protein envelope. This requires the protein to to derivatize free hydroxyl groups and then survive the alkaline hydrolysis required to liberate provide a planar surface having a high density of carboxyl groups and involucrin, with 20% of the lipids from the envelope. Pig stratum corneum, previously extracted exhaustively with glutamate residues, is a candidate for this role. The present study sought to determine a chloroform:methanol:1% acetic acid, was immersed in a solution of TIPS:pyridine: AgNO3 (4:2:1) conformation by which involucrin might present its glutamate residues in the required surface in acetone. After shaking overnight, the tissue was removed, rinsed with methanol, and then density and planarity. Computer modeling showed that if folded in a specific 01α-helical treated with 1 M NaOH in 95% methanol at 60°C for 1 h. The liberated lipids, recovered in conformation, involucrin could provide the needed array of carboxyl groups throughout the chloroform and analyzed by thin layer chromatography, contained two principal components, repeating consensus sequence (PEQQEGQLEL)n. Analogues were synthesized in which n ϭ 1, which were isolated by TLC. A sample of protein-bound 01ω-hydroxyceramide was isolated 2, and 3, producing peptides containing 10, 20, and 30 residues, in which the amino ends were from pig stratum corneum without prior treatment with TIPS and then fully derivatized with that blocked by acetylation and the carboxyl ends by amidation. All three peptides were soluble in reagent. NMR studies, including 2D COSY experiments, allowed the determination that of the buffer at pH 7, where their circular dichroic (CD) spectra indicated random coil conformations, two products from in situ derivatization, the more mobile on TLC (40% of the binary mixture) as expected for short peptides containing both proline and glycine residues. However, when had the 01ω-hydroxyl group free and must therefore have been attached to protein through that dissolved in trifluoroethanol, the 20- and 30-residue peptides produced CD spectra of 100% 01α- group. This result eliminates an alternative possibility in which all hydroxyceramide molecules helices, while the 10-residue peptide became insoluble. These results indicate that the proposed might have been bound through the 01ω-hydroxyl group while 60% also had the sphingosine 01α-helical conformations of the longer peptides may be stabilized by intramolecular hydrogen head group blocked in some way. The NMR results also indicated that the sphingosine 3-hydroxyl bonds and hydrophobic interactions, while the shorter peptide precipitates in TFE because group is always derivatized by TIPS and therefore is never involved in the binding to protein, a predominantly intermolecular interactions are available. The proposed 01α-helical conformation finding that was expected but previously unproven. places all of the glutamine residues in locations where they could cross-link with a substrate protein while also providing intramolecular hydrogen bonding.

315 316 Defining Size of Clonal Population of Keratinocytes in Normal (NN) Skin With Expansion of Protection of Human Skin Against Microbial Pathogens is Mediated by Antimicrobial Peptides Clonal Size in Pre-Psoriatic (PN) Skin S. Kippenberger, B. Traupe, J-M. Schroeder,* E. Christophers,* G. Sauermann and F. Wolf S. Chu, V. Chaturvedi, S. Smith and B. Nickoloff Department of Microbiology, Paul Gerson Unna Skin Research Center, Beiersdorf AG, Hamburg, Department of Pathology, Cardinal Bernardin Cancer Center, Loyola University, Chicago, Illinois Germany; *Department of Dermatology and Venerology, Christian-Abrechts-Universita¨t, Kiel, While it is postulated that a keratinocyte stem cell is at the epicenter of a clonal population of Germany cells derived from the stem cell rich follicular bulge that populate the overlying epidermis, the Human skin is constantly exposed to physicochemical and microbiological challenges from the exact location, distribution, and number of stem cells in human skin is unknown. Furthermore, environment. Apart from a mechanical barrier, provided by the skin surface, a microbiological it is unclear if PN skin is different from normal skin with respect to clonal populations barrier has been postulated to exist within skin. In addition to the already well known antimicrobial of keratinocytes. lipids, we were now able to demonstrate that also antimicrobial peptides with broad-spectrum To address these issues, 98 samples of various sizes were analyzed for clonality from a single activity against microbial pathogens are expressed in human skin. In addition to being expressed healthy female without psoriasis employing X chromosome inactivation patterns at the HUMARA in the epidermis, expression of such peptides could be localised to apocrine and eccrine sweat locus. Using this data base, biopsies of PN skin were compared/contrasted using the same glands, the hair follicle and sebaceous glands, indicating that exogeneous microbial challenges may HUMARA assay. DNA was isolated and clonality established by loss or significant decrease in play a role in inducing and regulating the expression of epidermal antimicrobial peptides. In either the maternal or paternal X chromosome following selected restriction enzyme digestion, addition to providing a mechanism for efficient control of microbial colonisation of human skin PCR amplification and gel electrophoresis. Thirty of 48 consecutive 2-mm punch biopsies were it could be demonstrated that disruption of epidermal integrity, e.g. by inflammatory processes clonal, whereas only four of 20 3-mm punch biopies, six of 17 4-mm punch biopsies, and no 5- following microbial invasion, resulted in a considerable increase in expression of epidermal mm punch biopsies revealed a clonal population of keratinocytes. Mantel-Haenszol Chi-square antimicrobial peptides. Similar alterations in expression could be obtained by stimulation of skin analysis revealed a p-value of 0.001, reflecting a strong trend in the probability of the presence of cell cultures using intact bacteria, bacterial cell wall components and a number of inflammatory a single clone of keratinocytes as related to the size of normal skin sampled. Interestingly, within mediators. The mechanisms of induction of such peptides will be disclosed, providing further the 48 2-mm punch biopsies removed in-contiguity there were at least 10 clonal changes as insight into the relevance of epidermal antimicrobial peptides for maintaining the antimicrobial reflected in maternal versus paternal switches in the sequential analysis. By contrast in five different barrier of human skin. PN biopsies of 4 or 5 mm size, all but one were clonal, and even an 8-mm punch biopsy contained a clonal population of keratinocytes. These results indicate that a clonal population of keratinocytes can be frequently detected in very small biopsies (2 mm), but that in NN skin overlapping clones are apparently present in larger (i.e. 4–5 mm) biopsies producing nonclonal patterns using the HUMARA assay. Since human hair follicles are approximately 1–2 mm apart, it would appear that a single stem cell in the follicular bulge is responsible for producing a clonal population of keratinocytes that is contained within a 2-mm punch biopsy. However, in PN skin, there may be clonal expansion of keratinocytes due to perturbation in epidermopoiesis and/or stem cell distribution/activity.

317 318 Unexpected Localization of the Zinc Finger Protein rfp to the Nucleolus of Keratinocytes Development of a Pig Living Skin Equivalent as an Animal Model to Study Skin Regeneration R. Livingston, B. Dale and K. Stephens Using Cultured Cells Departments of Pathology, Oral Biology, and Medicine, University of Washington, Seattle, Wash- V. Ronfard, J. Potzka, E. Govignon and J. G. Rheinwald ington Laboratory of Cell and Tissue Development, Organogenesis Inc., Canton, Massachusetts; Division The zinc finger protein rfp was identified as a putative intermediate filament-associated protein of Dermatology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts due to its ability to bind the keratin 5 head domain in the yeast two-hybrid system. Polyclonal Organotypic culture systems such as the living skin equivalent (LSE) have long been used in antibodies against rfp peptides recognized the predicted 58 kDa protein in western blots of foreskin research and more recently for therapeutic purposes (e.g. Apligraf). Xenografts on immune- epidermal extracts. Immunocytochemistry revealed that rfp was primarily in the nucleus of deficient, athymic mice and on humanized SCID mice have been used to study the biology and suprabasal cells of foreskin. Indirect immunofluorescence of cultured primary foreskin keratinocytes immunology of human LSE, but investigation of LSE biology in a normal recipient has been confirmed these results and further localized rfp to subnuclear bodies. Weak cytoplasmic signal difficult owing to the lack of a suitable animal model. Because of histologic similarities to human was also observed. Because rfp was reported to localize in HeLa cells to subnuclear POD structures skin, the pig has been recognized as an excellent animal model for studying cutaneous wound that are altered during carcinogenesis, double label immunofluorescence was used to investigate healing. We have sought to culture epidermal keratinocytes and dermal fibroblasts from pig skin the localization in keratinocytes. These data demonstrate that although keratinocytes have PODs, and to develop a pig LSE. In the past, derivation of a suitable cultured keratinocyte population rfp does not colabel with these structures, but rather with the nucleolus. Our results implicate rfp from pig was complicated by the presence of contaminating cell types. The use of sequential in novel keratinocyte nucleolar functions that indirectly could involve keratin filaments. thermolysin and trypsin digestion improved the yield and purity of proliferative keratinocytes from split-thickness pig skin biopsies. Keratinocytes isolated from fetal, newborn, and adult pig skin could be serially cultured on collagen-coated dishes with feeder fibroblasts of fetal pig fibroblast origin for Ͼ12 passages and 50 doublings. When cultured at the air–liquid interface on a collagen matrix containing human dermal fibroblasts, serially passaged pig keratinocytes formed a stratified, differentiated epidermis similar to that formed by cultured human keratinocytes. Fetal pig dermal fibroblasts were also able to contract a collagen gel and support epidermal histogenesis of pig keratinocytes to form an LSE suitable for grafting. These results establish that pig keratinocytes and fibroblasts can be extensively propagated in culture and used to form an LSE, thereby providing material for a normal animal model to study skin regeneration using grafts of cultured cells. 576 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

319 320 Neutrophil Gelatinase-Associated Lipocalin is Distinctly Induced in Keratinocytes Underlying Increased Rab11a Expression in Differentiating Primary Mouse Keratinocytes Parakeratotic Areas in Psoriasis R. Griner, J. Goldenring and W. Bollag L. Mallbris, A. Hultheń, M. Frohm-Nilsson, N. Borregaard and M. Ståhle-Ba¨ckdahl Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia Department of Dermatology, Karolinska Hospital, Stockholm, Sweden and Granulocyte Research Members of the Rab subfamily of small GTP-binding proteins are thought to regulate protein– Laboratory, Department of Hematology, Rigshospitalet, Copenhagen, Denmark protein interactions involved in vesicle targeting and fusion. Individual Rab proteins localize to NGAL, neutrophil gelatinase-associated lipocalin, is a 25-kDa protein initially identified in the distinct intracellular membrane surfaces. Specifically, Rab11a is highly expressed in apical vesicular specific granules of the neutrophil. The biologic functions of NGAL are un-known, but due to populations in numerous epithelia including: gastric mucosa, esophogeal mucosa, hepatocytes, its ability to bind small lipophilic substances and bacterial products, an immunomodulatory role renal collecting duct cells, and the intermediate differentiating layers of rabbit skin (Am J Physiol in the barrier defense has been suggested. Recent data have shown that NGAL is induced in 270:G515–G525, 1996). Because Rab 11a is highly expressed in differentiating keratinocytes in colon epithelium associated with diverse inflammatory conditions. In this context we have the rabbit, we sought to determine if Rab 11a expression was dependent on the differentiation examined the production of NGAL in skin in-flammation, and partly in contrast to the general status in primary cultures of mouse keratinocytes. Keratinocytes were induced to differentiate by upregulation reported in colon, we found a selective induction of NGAL mRNA and protein adding either 100, 250, or 1000 01µMCaϩϩ to the media for 24 h (control ϭ 25 01µMCaϩϩ) only in psoriasis. No NGAL was detected in contact allergy reactions, in atopic eczema, in lichen and the expression of Rab11a mRNA was examined by northern blot analysis. Although Rab11a ruber or in healthy interfollicular epidermis. In psoriasis, the NGAL expression was distinctly mRNA expression was increased by each of the higher concentrations of Caϩϩ, keratinocytes confined to the superficial one-third of the epidermis underlying parakeratotic areas and correlated induced to differentiate by 250 01µMCaϩϩ increased Rab11a mRNA most significantly (206% negatively with immunoreactivity for filaggrin, a marker for terminal differentiation. In neutrophils, Ϯ 44% versus control). Additionally, Rab11a mRNA expression was increased by inducing NGAL is covalently bound to 92 kDa gelatinase (MMP9), but in psoriasis we detected no signal keratinocyte differentiation with 1,25-dihydroxyvitamin D3. Initial examination of Rab11a protein or immunoreactivity for 92 kDa gelatinase in NGAL-positive cells. Thus, our present data expression by western blot analysis suggests that the increases in Rab11a mRNA are reflected in demonstrate the induction of NGAL, a potentially immuno-modulatory protein, in a spatially increases in Rab 11a protein. These results suggest that increased levels of Rab11a may be necessary distinct subset of psoriatic keratinocytes characterized by disturbed differentition. for specific vesicle targeting events during the intermediate stages of keratinocyte differentiation.

321 322 A New Method for Studying Epithelialization and Hair Follicle Formation from Cutaneous Cells The Polyunsaturated Fatty Acids Linoleic Acid (18:2, n-6), Arachidonic Acid (20:4, n-6) and of the Mouse Lignoceric Acid (22:6, n-3) Inhibit the Arachidonic Acid Conversion Seen In Essential Fatty Acid R. J. Morris, K. A. Tryson and K. Q. Wu Deficient (EFAD) Keratinocytes The Lankenau Medical Research Center, Wynnewood, Pennsylvania C. L. Marcelo and W. R. Dunham We have developed a grafting assay for epithelialization and hair follicle formation requiring 10- Department of Surgery and Biophysics Research Division, University of Michigan, Ann Arbor, fold fewer cells than previously reported methods. This technique uses de-epithelialized rat Michigan tracheas, the ends of which are sealed with hemoclips and stretched onto thin Teflon tubing. The In vitro EFAD keratinocytes are established by growth in serum-and fatty acid-free medium. EFAD tracheas are inoculated with 1 01ϫ 106 fresh or cultured keratinocytes and either 1 01ϫ 106 fresh cells grow rapidly, have elevated levels of the monoene fatty acids 16:1 and 18:1 and minimal newborn fibroblasts or cultured dermal papilla cells. The inoculated tracheas are implanted amounts of 18:2-and 20:4-fatty acids. The EFAD cells convert 18:2–20:3 and to 20:4-fatty acids subcutaneously into athymic mice for 2 wk to allow epithelialization or for 4 wk to study the (not seen in in situ epidermis). Polyunsaturated fatty acid (PUFA) addition to the medium formation of hair follicles. Fresh or cultured cutaneous epithelial cells from adult mice or cultured normalizes the fatty acid levels to tissue values, decreases cell proliferation and lowers the monoene stem cell colonies formed a two-layered epithelium composed of a basal and suprabasal layer. content. GC, HPLC, and Radioactive Labeling analysis were used to study the conversion of 14 Epidermal cells from mice initiated with 200 nmols of 7, 12-dimethylbenz-[a]anthracene formed C-18:2–20:4 by the PUFA-normalized cells. Cultures grew in EFAD or PUFA-supplemented a normal appearing epithelium, whereas cells from initiated and promoted mice formed dysplastic media for 2–3 passages. Three fatty acid media were tested: 1) medium ϩ 10 mM 18:2 and 5 exophytic foci. Formation of hair follicles and sebaceous glands was observed when fresh or mM 20:4, 2) medium ϩ 10 mM 22:6, and 3) medium ϩ 10 mM 18:1. The cells were labeled cultured epithelial cells from newborn mice were inoculated together with newborn dermal with 14 C-18:2 and were harvested after ddd, 1, 2, 4, 8 and 24 h. Conversion of 14 C-18:2 via fibroblasts or a dermal papilla cell line. Fresh cutaneous epithelial cells from adult mice have not 14 C-20:3 to the 14 C-20:4-fatty acid occurred at all time points in the EFAD cells and cells grown yet been observed to form hair follicles in this assay. This technique gives reproducible results and in 10 mM 18:1 medium. The two PUFA-media abolished this conversion. EFAD cells were is easy, requiring no special surgical skills. This technique will be useful for studying stem cells switched to the PUFA-media for 24-h prior to radiolabeling. Analysis showed that the PUFAs from the cutaneous epithelium of the mouse as well as for the study of factors modulating the were present in the cell phospholipids and that the PUFAs partially reduced the conversion of 14 development of hair follicles when the number of inoculating cells is limiting. C-18:2–20:4. Cyclohexamide, a protein synthesis inhibitor at 0.5 mg per ml eliminated this enzyme activity. The data shows that (i) EFAD cells express the desaturase enzymes that convert saturated fatty acids to monoenes, and 18:2–20:4; (ii) restoration of cellular PUFA levels obliterates this activity; (iii) and the desaturase activity is obliterated by inhibition of protein synthesis. We conclude that while epidermal cells normally do not express the enzymes responsible for the conversion of 18:2–20:4, under certain, potentially pathologic conditions, this pathway can be vigorously expressed by keratinocytes.

323 324 The Effects of Tropospheric Ozone on a Human Epidermal Skin Model (Epiderm) Epidermal PAI-2 Expression and Endogenous Cleavage during Keratinocyte Differentiation and S. U. Weber, A. Tavakkol,* Z. Nabi,* S. Jothi, T. G. Polefka* and L. Packer after Barrier Disruption Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, B. Risse, N. Chung, S. Kim, R. Lavker, J. Leyden, D. Saunders,* M. Baker† and P. Jensen California; and *Personal Care Products, Colgate-Palmolive Co., Piscataway, New Jersey Department of Dermatology, University of Pennsylvania, Philadelphia, Pennsylvania; *Department Recent reports suggest that exposure to tropospheric ozone (O3) poses an oxidative stress to of Biological Sciences, Univ of Wollongong, Australia; and †Gynaecological Cancer Research murine skin. So far, no data is available on human skin. Monolayers of human keratinocytes Center, Royal Women’s Hospital, Melbourne, Australia cannot serve as a model of human skin for O3 exposure, since O3 mainly reacts within the Plasminogen activator inhibitor type 2 (PAI-2) has been hypothesized to play an intracellular role stratified stratum corneum (SC). We chose a organotypic model of human epidermis (Epiderm) late in epidermal differentiation, during cornified envelope formation, perhaps through interaction with a differentiated SC to study the effects of environmental O3 exposure. with an unknown cytoplasmic proteinase. Cleavage of PAI-2 in its reactive site loop by interaction The tissue was exposed to 5 p.p.m. O3 for 2 h, which did not affect the tissue viability, as with a proteinase leads to the generation of a more stable or ‘‘relaxed’’ conformational state. We measured by LDH release. Mock exposure to air only for 2 h was used as control. Glutothione have utilized a monoclonal antibody that specifically recognizes relaxed PAI-2 (PAI-2r) to (GSH), Vitamin C, uric acid, and Vitamin E as antioxidants, and malondialdehyde (MDA) as an address whether epidermal PAI-2 does indeed interact with an endogenous proteinase. Using indicator of lipid peroxidation were analyzed by HPLC in whole tissue. Uric acid levels were not immunohistochemistry, we found that normal human epidermis contains PAI-2r, which is localized changed while GSH increased by 40% (p Ͻ 0.05). Vitamin E, the major lipophilic antioxidant, predominantly around the periphery of the granular cells. This localization pattern is similar to was not depleted by this dose of O3. Vitamin C was not detected in the tissue culture. No that observed with an antibody that detects all forms of PAI-2 and indicates that at least some significant MDA production was observed. To study the effects of O3 on exogenous Vitamin E, PAI-2 in the granular layer of normal human epidermis has been cleaved by endogenous proteinase. µ 20 l of a 0.5% preparation of a-tocopherol in PEG 400 was added to the wells 2 h prior to O3 To determine whether perturbation alters epidermal expression patterns of PAI-2, we applied 1% exposure. This pretreatment increased Vitamin E levels by 2.5 fold. Ozone exposure depleted this SDS or 0.5% tretinoin under occlusion to the skin of normal volunteers 24 h prior to biopsy. added Vitamin E by 30% (p Ͻ 0.001). However, the Vitamin E remaining in the skin was still With either antibody (i.e. to all forms of PAI-2 or to PAI-2r), the staining intensity in the treated significantly higher than the endogenous portion (p Ͻ 0.001). epidermis was increased as compared to nontreated control epidermis; furthermore, PAI-2 in the These results indicate that specific cellular changes are induced by a single acute exposure to O3 treated epidermis was not limited to the granular layers, but rather was also detectable in several in the organotypic skin model. Pathophysiological consequences remain to be investigated. spinous layers. These data suggest that disruption of the barrier leads to induction of epidermal PAI-2, a finding that may reflect a function of this proteinase inhibitor as part of a well- coordinated, protective response to restore epidermal homeostasis. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 577

325 326 Distinct Retinyl Esters Loading in Epidermis after Topical Application of Retinaldehyde and Identification and Regulation of Secretory Phospholipase A2 in Non-Confluent Primary Human Retinoic Acid on Hairless Mouse Skin Keratinocytes: Implications in Wounding Healing O. Sorg, L. Didierjean, P. Carraux, G. Siegenthaler and J. Saurat K. Rys-Sikora, R. Konger, J. Schoggins, R. Malaviya* and A. Pentland Department of Dermatology, University Hospital, Geneva, Switzerland Department of Dermatology, University of Rochester, Rochester, New York and *Wayne Hughes Natural retinoids such as all-trans-retinoic acid (RA) and -retinaldehyde (RAL) are currently used Institute, St. Paul, Minnesota as topical agents in humans. The aim is to deliver, either directly (RA) or indirectly (RAL), the Secretory Phospholipase A2s (sPLA2s) are thought to be key enzymes in wound healing. The ligand for nuclear receptors, i.e. RA, into the skin. We have now evaluated the effect of these production of fatty acids and subsequent cyclooxygenase (COX)-dependent prostaglandin E2 topical retinoids on the content of vitamin A under its storage form, i.e. all-trans-retinol (ROL) (PGE2) production are associated with multiple wounding events including cell proliferation, cell and ROL esters (RE). We determined by RP-HPLC the concentrations of epidermal (heat- migration, and bacterial and fungal killing. Despite their importance, sPLA2s in human keratinocytes separated) ROL and RE following a 7-day application of 0.05% RA or RAL on the back of has been poorly characterized, In in vitro wounding experiments, the production of PGE2 was hairless mice. Vehicle-treated epidermis contained, in pmol/g wet weight: RA, Ͻ 15; ROL, 152 biphasic suggesting coordinated regulation of PLA2s and COX enzymes in epithelial repair. Ϯ 20; RE (sum of ROL esterified with long chain fatty acids, i.e. C14-C20), 1102 Ϯ 85. As Primary human keratinocytes grown in nonconfluent (NC; Ͻ 30% confluent) conditions behave compared to vehicle-treated, RAL-treated epidermis contained 6.2-fold more ROL and 12.3- similarly to ‘‘activated’’ keratinocytes in c wound; they are highly proliferative, migratory, and fold more RE. In contrast, after topical RA, epidermal ROL was decreased by 30%, while have an elongated morphology. NC cultures released 10-fold more PGE2 and have higher epidermal RE concentrations were increased by 2.3-fold. On the other hand, epidermal RA expression of both COX-1 and COX-2 than confluent cultures. Mass spectrometric analysis of concentrations after a topical treatment with RAL and RA were 226 nM and 1487 nM, respectively. supernatants taken from NC cultures showed a 2:1 ratio of arachidonic acid (AA):linoleic acid Since RA cannot be converted into ROL and RE, the decrease of ROL and the increase of RE (LA) release indicating the presence of both cytosolic PLAZ (cPLA2) and sPLA2 activities in NC after topical RA indicates this occurs through increased activity of lecithin:retinol acyltransferase cells. RT-PCR analysis of sPLA2 isozymes indicated the presence of both type IIA and type V (LRAT), an enzyme that esterifies ROL with long chain fatty acids (Shimada et al., Arch Biochem sPLA2. In immunoblot experiments, no difference in cPLA2 protein expression was observed in Biophys 344:220–227, 1997). In contrast, RAL, which can be either reduced into ROL, or NC and confluent total cell lysates, whereas there was a 3-fold increase in sPLA2 protein levels oxidised into RA, would load the skin with RE by both modulating the activity of LRAT and in NC cell lysates. Although sPLA2 was also detected in basal NC supernants, a high amount of producing a substrate for this enzyme, which explains the higher content in ROL and RE after protein appears to be cell-associated. sPLA2 levels were diminished by 24 h treatment with topical RAL. pertussis toxin (20 ng per ml) suggesting Gprotein-dependent regulation. Thus, increased sPLA2 expression and activity play an important regulatory role in the activated keratinocyte and understanding its regulation may bring some insight into their multifunctional role in wound repair.

327 328 Characterization of Neuropeptide Receptor mRNA Expression in Normal Human Keratinocytes Expression of Integrin Molecules on Human and Mouse Keratinocytes and the Murine Keratinocyte-like Cell-Line PAM 212 L-J. Hu, J. S. Wallner and C. L. Reardon E. Miranda, A. Tabaee, H. Ozawa, K. Campton and R. D. Granstein Department of Dermatology, University of Colorado Health Sciences Center, Denver, Colorado Weill Medical College of Cornell University, New York, New York CD11a, CD11b and CD11c (01αL, 01αM and 01αX, respectively) together with CD18 (01β2) The epidermis is innervated and several neuropeptides have been shown to modulate aspects of protein chains combine to form heterodimeric cell surface integrin molecules. These integrin keratinocyte physiology. Therefore, we investigated the expression of mRNA for neuropeptide adherence proteins, CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1 or CR3) and CD11c/CD18 receptors in both normal human epidermal keratinocytes (HEK) from pooled neonatal foreskin (p150,95 or CR4) are not thought to be keratinocyte cell surface markers. The past focus of and in PAM 212 cells. Total RNA was isolated from HEK and reverse transcribed (RT) with keratinocyte interactions with cells has been on the expression of ICAM molecules on keratinocytes oligo-dT single stranded DNA to select for mRNA. For PAM 212 cells, mRNA was purified with little attention on the expression of ICAM ligands. With the methods of fluorescent and cDNA was reverse transcribed. Using cDNA from both cell types, primers designed from monoclonal antibody staining of cells and flow cytometry, we have found that keratinocyte cell published sequences as well as previously published primers were used to amplify cDNA fragments lines constitutively express integrin molecules. Both CD11a and CD11c are expressed by the coding for various neuropeptide receptors. ublished primers were used to amplify cDNA fragments mouse keratinocyte lines, PAM212 and XB-2 cells, and by the human keratinocyte lines, A431 coding for various neuropeptide receptors. DNA was al keratinocytes (HEK) from pooled neonatal and HaCaT. In contrast, CD11b expression showed species disparity with no CD11b expression foreskin and in PAM 212 cells. Total RNA was isolated from HEK and reverse transcribed (RT) on the mouse keratinocytes but positive expression by the human keratinocytes. Why there are with oligo-dT single stranded DNA to select for mRNA. For PAM 212 cells, mRNA was purified species differences in CD11b expression by keratinocytes is unclear. CD11b, a classical marker for and cDNA was reverse transcribed. Using cDNA from both cell types, primers designed from macrophages, is commonly expressed on these cells but not on many other cell types. Thus, published sequences as well as previously published primers were used to amplify cDNA fragments CD11b expression by the human keratinocyte lines is unusual. CD18, the 01β chain of the coding for various neuropeptide receptors. Thirty-five cycles of PCR were performed for the CD11/CD18 class of integrin molecules, is expressed by mouse and human keratinocytes as human cells and 40 cycles for the PAM 212 cells. PCR products of the neuropeptide receptor expected. Like their cell line counterparts, primary human keratinocytes also express these integrin cDNA fragments were identified based on expected size and restriction enzyme analysis. In HEK, molecules but only after cell stimulation with agents, such as PMA or LPS. As evidence exists in products of the expected size were obtained for somatostatin receptor (SSTR) subtypes 1, 2 and other cell systems that the integrin molecules serve to transmit signals intracellularly, the interactions 5 and the vasoactive intestinal peptide-1 receptor (VIP-1R). The presence of SSTR-2, SSTR-5 of integrin molecules on keratinocytes with the integrin ligands on other keratinocytes may lead and VIP-1R [pituitary adenylate cyclase activating polypeptide (PACAP)-II receptor] were to interkeratinocyte stimulatory signaling in the epidermis. subsequently confirmed via restriction enzyme analysis. In the PAM 212 cell line, products of the expected size were obtained for SSTR-1, 2, 3, 4 and 5; the galanin receptor and PACAP-I, II and III receptors. The SSRT-2 and 4 receptors and the galanin receptor were confirmed with restriction enzyme analysis. Keratinocytes appear to express receptors for several neuropeptides, suggesting regulation of keratinocyte physiology by products of nerves.

329 330 Anti-Apoptotic Gene Expression in Human Keratinocytes Transgilutaminase Reactivity of Human Involucrin Y. Shellman, G. Bellus, I. Maxwell and . DA Norris A. Lambert, M. Ekambaram, N. Robinson and R. Eckert Department of Dermatology, University of Colorado Health Science Center, Denver, Colorado Physiology & Biophysics, Dermatology, Biochemistry, and Oncology, Case Western Reserve The skin, and especially the epidermis, is constantly at risk for cell death triggered by ultraviolet University School of Medicine, Cleveland, Ohio radiation and toxins, by oxidant stress and by toxic products of the immune response. In this toxic Involucrin (hINV) is assembled into cornified structures via formation of transgiutaminase- environment, defenses against apoptosis would seem to be essential to maintain the integrity of dependent interprotein 01ε-(01γ-glutamyl)lysine bonds. The hINV sequence includes 150 Q key cell populations. A better understanding of defenses preventing apoptosis in keratinocytes is residues that could function as potential sites. crosslink formation. The present studies were therefore very important. Endothelial cells express a variety of antiapoptotic proteins constitutively, designed to evaluate the extent to which hINV can function as a TG substrate under optimal and also express unique endothelial cell specific antiapoptotic proteins after stimulation with conditions and in the absence of other substrates. Incubation of 20 µM rhINV with 0.002 units primary cytokines IL-1 or TNF-01α. We questioned whether keratinocytes expressed unique transgiutaminase results in formation of 4–5 isopeptide bonds per hINV molecule. When 4 mM Bcl-2 homologs constitutively or following cytokine stimulation. Our purpose was to determine 14C-putrescine is present in the reaction, 10 Q residues are labeled per hINV molecule, and at the level of expression of known antiapoptotic proteins in human keratinoicytes, and to use RT- higher 14C-putrescine and TG levels 48 Q residues are found to be TG reactive. Isotope PCR (reverse transcription polymerase chain reaction) to identify unique keratinocyte-specific distribution and sequence analysis suggests that the labeling sites are distributed throughout the apoptotic gene expression. Using degenerate primers complementary to two conserved regions of protein. In contrast to in vivo results indicating the selective crosslinking of specific residues, our Bcl-2 family members, we searched for Bcl-2 homolog gene expression. These searches were present results show that many residues, distributed throughout, the protein, can function as TG performed on cultured human keratinocytes without any triggers and following TNF-a stimulation. substrates. These results show that involucrin can be labeled at high crosslink densities, and suggest In cultured human keratinocytes, the only messages identified for Bcl-2 homologs were for Bcl- that, in vivo, other factors other than hINV structure must influence the selection of sites of xL and Mcl-1. This correlated with Western immunoblots which showed that Bcl-xL and Mcl-1 crosslink formation. were the most prevelent antiapoptotic proteins. No unique Bcl-2 homologs were identified in baseline or TNF-01α stimulated cultures. Levels of Bcl-2 detected by immunoblot were low in cultured keratinocytes, but were evident by immunohistochemistry in keratinocytes in skin biopsies and in organotypic cultures containing keratinocytes but not melanocytes. We found that the antiapoptotic defenses in keratinocytes are provided by expression of the proteins Bcl-2, Bcl-xL and Mcl-1, which are differentially controlled. No unique Bcl-2 homologs were identified using RT-PCR with degenerate primers. 578 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

331 332 Transglutaminase-Dependent Covalent Modification of S100 Proteins Possible Roles of Peptidylarginine Deiminase During the Terminal Stage of Normal and Abnormal A. Lambert, N. Robinson and R. Eckert Epidermal Differentiation Physiology & Biophysics.Dermatology, Biochemistry and Oncology, Case Western Reserve M. Mizoguchi, Y. Kondo, M. Manabe,* T. Senshu† and H. Ogawa University, Cleveland, Ohio Department of Dermatology, Juntendo University School of Medicine, Tokyo, Japan; *Department S100 proteins are an important family of calcium-binding proteins, located within the epidermal of Dermatology, Akita University School of Medicine, Akita, Japan; and †Department of Bioactivity differentiation complex (EDC) on 1q21, that function to transmit calcium-dependent signals. Regulation, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan Upon binding calcium, S100 proteins undergo a conformation change that permits S100 protein Peptidylarginine deiminase (PAD) is the enzyme which converts protein arginine residues to binding to specific target proteins. This activated complex produces the biological response. citrulline residues. A recent study has shown that the major deiminated proteins in human Keratinocytes express eight distinct S100 proteins including S100A2, S100A4, S100A6, S100A7, epidermis are partially degraded keratin K1 and that the minor components are derived from S100A8, S100A9, S100A10, and S100A11. We previously described S100A11 is a type 11 filaggrin, trichohyalin, and keratin K10. To explore further the role of protein deimination in transgiutaminase (TG2) substrate, and hypothesized that TG-dependent covalent labeling may epidermis, we attempted to identify the deiminated proteins in cultured epithelial cells after modify S100A11 function. We now expand these studies and identify type 1 transgiutaminase activation of endogenous PAD by ionomycin treatment in the presence of 2 mM calcium. We (TG1) and TG2 reactive amino acid residues in S100A11, S100A10, and S100A7. S100A11, but found that a 70-kDa nuclear protein is the major deiminated protein in these cultured cells and not S100A10 or S100A7 form crosslinked multimers. Moreover, although they have a similar that the transfection of a PAD cDNA causes deimination and disintegration of the nucleus in domain structure, S100 proteins are differentially reactive TG substrates (S100A10 Ͼ S100A11 Ͼ transfected cells. In addition, we examined the localization of deiminated proteins in hyperplastic S100A7). Each S100 protein contains a single reactive Q residue in the amino (S100A7-Q5, epidermis. In normal epidermis, deiminated proteins were localized mainly in the cytoplasm of S100A10-Q4) or carboxyl (S100A11-Q103) terminus. TG1 and TG2 label the.same residue, Q103, the lowermost cornified layer, whereas in parakeratotic cornified cells, these proteins were found in S100A11. In addition, immunological studies show that S100A11, S100A10 and S100A7 are mainly in the nucleus. These findings led us to speculate that PAD might induce nuclear disassembly components of cultured keratinocyte and foreskin cornified envelopes. Our studies suggest that during normal epidermal differentiation. However, during abnormal epidermal differentiation, it transglutaminase-dependent modification regulates the function of the S100 proteins. Further appeared that PAD acts mainly on 70 kDa nuclear protein, but not on K6/K16 in parakeratotic studies are underway to understand the role of this EDC-localized family of proteins. cornified cells, and that the protein deimination is not followed by other mechanisms responsible for nuclear disassembly.

333 334 The Effect of Dimethyl Sulfoxide(DMSO) Exposure on the Viability of Keratinocytes Nicotine-Enhanced Epithelial Differentiation in Reconstructed Human Oral Mucosa In Vitro K. Seo, J. Han and H. Eun J. Han, K. Seo, O. Kwon, J. Chung, D. Suh, K. Cho and H. Eun Department of Dermatology, Seoul National University College of Medicine, Seoul, Korea Department of Dermatology, Seoul National University College of Medicine, Seoul, Korea. Dimethyl sulfoxide(DMSO) is widely used as a cryoprotectant of keratinocytes cryopreservation. Oral mucosal keratinocytes represent the cells that first encounter tobacco components. Therefore, It has been generally accepted that the introduction and removal/dilution of DMSO can be a tobacco-induced abnormal alteration of the mucosal keratinocytes may contribute to the develop- source of lethal osmotic stress to a variety of mammalian cells. However, there have been no ment of oral white lesions. Nicotine is an ingredient of all tobacco products and pharmacologically reports on the effect of DMSO exposure without cryopreservation on the viability of keratinocytes. the most active component of tobacco smoke. To clarify the effects of nicotine on the keratinization Our purpose was to evaluate the toxicity of DMSO to keratinocytes and to establish proper of oral mucosal and epidermal keratinocytes, we reconstructed artificial buccal mucosal and skin protocol dealing with DMSO by means of controlled factorial experiments. We conducted the equivalents using keratinocytes and fibroblasts from noncornifying buccal mucosa and adult factorial experiments using the condition of the amount of exposure time(5, 15, 30, 60 min), foreskin, respectively. The effect of nicotine on keratinization was assessed with morphology, concentration of DMSO(10%, 20%, 30%) used, temperature of mixture (at 4°C and at room immunohistochemistry and immunoblotting. Long-term treatment with nicotine for 2 wk temperature) and the way of introduction(single-step, step-wise) and removal/dilution(single-step, enhanced in a dose-dependent manner the expression of differentiation-specific proteins of oral step-wise) of DMSO. This experiment was performed in quadruplicate using keratinocytes of mucosal keratinocytes on living oral mucosal equivalent and epidermal keratinocytes on living second passage from human foreskin and the trypan blue test was used to check the viability skin equivalent, respectively. The effect of nicotine on the cell viability was measured by of keratinocytes the MTT([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]) assay. Oral mucosal Our results showed that single step addition and removal/dilution of DMSO is significantly keratinocytes showed a higher resistance to nicotine toxicity than epidermal keratinocytes. Our superior to step-wise addition and removal/dilution, which was against to general expectation results suggest that nicotine stimulates differentiation of both mucosal and epidermal keratinocytes that rapid osmotic change can render more damage to cells than slow change. We also found that and this nicotine-induced abnormal differentiation may be associated with the development of there is no difference of DMSO toxicity to keratinocytes between handled at 4°C and room oral white lesions. temperature (20–24°C) and that DMSO is toxic to keratinocytes after only 5 min short-term exposure at 10% concentration. However, there was no significant reduction of viability of keratinocytes with increased concentration of DMSO and increased exposure time up to 1 h. These results suggest that we should do single-step introduction and removal/dilution of DMSO when using keratinocytes and that there is no need to proceed the cryopreservation of keratinocytes at ice.

335 336 Phytosterol Sulfate, a New Cholesterol Sulfate Homologue, Induces Human Keratinocyte Vitamin C Stimulates Sphingolipid Synthesis in Submerged Human Keratinocyte Cultures Differentiation And Keratin Synthesis Y. Uchida, M. Behne, D. Quiec, P. M. Elias and W. M. Holleran N. Domloge, S. Marguerie, A. Geffard, K. Golz and L. Zastrow Department of Dermatology, UCSF & Dermatology Service & Research Unit, VA Medical Department ofof Dermatology and Skin Care Research, Coty International Research and Center, San Francisco, California Development Center, Monaco Although cultured keratinocytes (CHK) differentiate in response to specific stimuli, the level and Keratinocyte differentiation and skin keratin layer functions are of great importance in skin biology. spectrum of ceramides (Cer) produced, especially in submerged culture systems, do not achieve Phytosterol sulfate (PS), is a new molecule that is developed from vegetables by semisynthesis those seen in vivo. Ponec et al. recently demonstrated that vitamin C supplementation (Vit C) method to enhance cell differentiation. Using various concentration and treatment times we stimulated an increase in specific ceramide (Cer) species, including glucosylceramide (GlcCer), in studied the effects of PS on cultured human keratinocytes, fibroblasts as well as organ cultures of a lifted culture system (J Invest Dermatol 109:348, 1997). Here, we used submerged CHK (switched human skin. In addition, comparative studies between the PS and Cholesterol sulfate (CS), an to DMEM/Ham’s F-12; 2:1, vol/vol; with 10% FBS at confluence) to determine whether important molecule for cell differentiation and barrier function, were conducted. Light microscopy alterations in sphingolipid content occur with Vit C, and to delineate the biochemical step(s) that studies demonstrated that cultured keratinocytes slowed proliferation and became more differenti- are enhanced/altered. Although total lipid content remained unchanged, Vit C (50 01µg per ml ated after PS application at 1% for 24 h. Specific immunostaining studies showed that PS enhances for 9 d postconfluence) increased both the total GlcCer and Cer, but not sphingomyelin, content the expression of differentiation markers such as Involucrin, and keratin 6 and 10 after 24 h of of submerged cells. Specific Cer and GlcCer species were preferentially enhanced (most notably, PS application; the maximum effect was found with 1% PS after 48 h. These results were Cer 2, Cer 4 [6-OH-AcylCer] & Cer 5 [01α-OH-Cer], but also Cer 6 and Cer 7; and three confirmed by immunoblotting studies of involucrin and keratins in treated and untreated GlcCer species: AcylGlcCer, GlcCer-B and GlcCer-D), while AcylCer and Cer 3 remained keratinocytes. Interestingly, studies of adhesion molecule expression in human keratinocytes unchanged. The incorporation of [14C]-acetate only into each of the corresponding GlcCer and demonstrated an enhanced staining of 01α301β1, 01β1 and 01α6, 24 h after PS application. To Cer fractions also was elevated. The N-acyl fatty acids of the total Cer and GlcCer fractions confirm PS effect on cell protein modulation, c-AMP dosage studies were performed on cultured revealed increased 01α-OH- and 01ω-OH-fatty acids, suggesting a preferential increase in human keratinocytes and fibroblasts. The results demonstrated an increase of total c-AMP in PS hydroxylated Cer and GlcCer species. The amount of 01ω-OH Cer, covalently bound to cornified treated cells with 1% PS for 24 h. These results were confirmed by studies of human skin in envelope protein (i.e. lipid-bound envelope), was increased with Vit C, while the formation of organ culture. Briefly, human skin samples were treated with PS, CS and placebo formulae for cornified envelope protein was unchanged. Finally, while Cer synthase activity was increased 24 and 48 h, then studied by H&E and immunostaining methods. PS application onto the skin (2.32-fold, p 01ഛ 0.01), both serine palmitoyltransferase and GlcCer synthase activities remained showed superior results to CS, demonstrating an improved, more moisturized, enriched skin unaltered. These results demonstrate that Vit C induces significant increases in selected Cer and barrier keratin layer and better conservation of the skin structure. These studies demonstrate that GlcCer species, even when CHK are grown under submerged conditions; and that these changes the new Phytosterol sulfate molecule plays an important role in enhancing cell differentiation. PS reflect both alterations in N-acyl fatty acid hydroxylation and the activity of specific Cer may alleviate various skin, hair and nail conditions and be of utility in cosmetology. synthetic enzymes. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 579

337 338 Langerhans Cells Express Matrix Metalloproteinase-9 in the Human Epidermis Dendritic Cell (DC) Maturation by Small Fragments of Hyaluronan (HA) Involves a Highly H. Uchi, S. Imayama, Y. Kobayashi and M. Furue Specific Nf-01κB and TNF01α-Dependent Pathway Department of Dermatology, Faculty of Medicine, Kyushu University, Fukuoka, Japan; Skin Care C. C. Termeer, U. Voith, H. Pahl,† C. Fieber,‡ S. Dichmann, J. Norgauer, E. Scho¨pf, P. Prehm* Division, R&D Headquarters, Sunster Inc., Asahi-machi, Takatsuki, Osaka, Japan and J. C. Simon Recent studies have shown that some cytokines regulate the migration of Langerhans cells from Department of Dermatology University of Freiburg, *Department of Biochemistry University of the epidermis, but the mechanism of this migration still unclear. We previously showed that the Muenster, † Institute for Tumor Biology, Freiburg, ‡ Institute of Genetics, Karlsruhe, Germany epicutaneous application of haptens stimulates the secretion of matrix metalloproteinase (MMP)- The glycosaminoglycan HA, build from repeating units of N-acetyl-D-glucosamine-UDP, is a 9 from Langerhans cell-enriched murine epidermal cells. MMP-9 can degrade type IV collagen major component of the cutaneous extracellular matrix. HA exists physiologically as a high that is one of the main components of the basement membrane. molecular weight polymer (HMW-HA), but is cleaved into lower molecular weight fragments We evaluated the normal skin specimens obtained from patients who underwent excisions of (sHA) at sites of inflammation. Here, we show sHA, generated from HMW-HA by enzymatic benign tumors. The specimens were cut into 3 01ϫ 3 mm pieces and incubated in RPMI-1640 digestion and gel column separation to potently induce maturation of human DC generated from containing 1 01ϫ 10–5 M monensin to accumulate intracellular enzymes. Some pieces were then peripheral blood progenitors as demonstrated by comparative analysis of immunophenotype, treated with dispase to obtain the epidermal sheets. The others were frozen and 30 µm-thick cytokine secretion, T-cell stimulatory function, chemokine receptor expression and chemokine cryostat sections were prepared. Both the sheets and the sections were incubated with monoclonal induced migration. We questioned, whether such DC-maturation is specifically induced by the mouse antihuman MMP-1, Ϫ2 and Ϫ9 antibodies. basal component of HA N-acetyl-D-glucosamine-UDP. To address this issue a number of We observed numerous MMP-9 positive dendritic cells to be present in the lower epidermis and structurally related substances were tested for their ability to stimulate DC-maturation including MMP-9 was present in the granules in their cytoplasms. Double labeling with anti-MMP-9 UDP-fucose, UDP-galactose, N-acetyl-D-galactosamine-UDP, and chondroitinsulfate. Import- antibody and anti-CD1a antibody demonstrated that the MMP-9 positive cells to be consistently antly, only N-acetyl-D-glucosamine-UDP dose-dependently induced DC-maturation, including positive for CD1a. These findings indicate that they are Langerhans cells. MMP-1 was expressed upregulation of B7-1, B7-2, CD83 and HLA-DR, a downmodulation of CD115, and a markedly by spinous and basal keratinocytes, but not by Langerhans cells. Dermal macrophage-like cells enhanced TNF01α-release comparable to sHA preparations, generated from HMW-HA. Since were also positive for MMP-1. MMP-2 positive cells were not detected in the epidermal sheets. TNF01α was described to be of principal importance for DC-maturation, the TNF01α-release The presence of MMP-9 in the CD1a positive cells in the epidermis supports the hypothesis that in response to sHA-treatment was studied in detail. Kinetic analysis revealed TNF01α m-RNA MMP-9 participates in the migration through the basement membrane. transcription rates to increase at 3 h and TNF01α protein secretion to peak at 12 h after sHA- stimulation, respectively. Moreover, mobility shift assays (EMSA) documented NF-01κB-activation 2 h after sHA stimulation, indicating that sHA-induced TNF01α-release results from nuclear translocation of NF-01κB. Finally, addition of a neutralizing soluble TNF01α-R1 prevented all sHA-induced maturational events in DC. Taken together, these results indicate that sHA-induced maturation of human DC involves a highly specific, Nf-01κB- and TNF01α-dependent pathway.

339 340 Osteopontin (OPN) Induces Epidermal Langerhans Cell and Dendritic Cell (DC) Migration. IL-18 and IL-12 Synergistically Upregulate IFN01γ Production by Murine Dendritic Epidermal J. Weiss, C. Maier, T. Ahrens, S. Kon,† M. Maeda,† H. Hotta,* E. Scho¨pf, T. Uede* and J. Simon T Cells (DETC) Department of Dermatology, University of Freiburg, Germany; *Institute of Immunological M. Sugaya, K. Nakamura and K. Tamaki Science, Hokkaido University, Japan; †Immuno-Biological Laboratory, Fujioka, Japan Department of Dermatology, University of Tokyo, Tokyo, Japan Following antigen capture immature DC leave peripheral organs and migrate to regional lymph Recent studies indicated that interleukin-18 (IL-18) was produced by murine keratinocytes and nodes to stimulate naı¨ve T lymphocytes. We recently described that differential expression of Langerhans cells. In this study, we investigated whether or not IL-18 and interleukin-12 (IL-12) CD44 isoforms is critical for DC migration and DC mediated induction of contact hypersensitivity upregulate IFN01γ production by murine dendritic epidermal T cells (DETC). α β in vivo. OPN a ligand for CD44 and 01 v01 3 integrin is a secreted glycoprotein with multiple Murine DETC were purified by the panning method (percentage of purity was always over 95%) functions during tissue repair and immune responses, OPN is secreted by and acts as a and 2 01ϫ 105 DETC were incubated with or without IL-12 and/or IL-18. IFN01γ concentrations chemoatractant for macrophages. To test whether OPN affects DC motility we performed of culture media were measured by ELISA method. Cytoplasmic mRNA was isolated from 1 modified Boyden chamber assays. These revealed that OPN dose-dependently induced DC 01ϫ 106 freshly purified and cultured DETC and the semiquantitative reverse-transcriptase migration. This effect could be blocked by mAbs against N-terminal CD44 epitopes. Further, polymerase chain reaction (RT-PCR) was performed. Viability of cultured DETC was calculated DC generated from CD44-/-mice showed reduced migratory efficiency towards OPN. To by staining cultured DETC with trypan blue stain. confirm that OPN affects DC trafficking in vivo we injected OPN subcutaneously into the pinnae IL-18 and IL-12 synergistically upregulated IFN01γ production by DETC at the protein and of mouse ears. Emigration of Langerhans cells was strongly induced by OPN, but not by mRNA levels and increased the survival rate of DETC during culture. This study suggests that appropriate control proteins. In an in vivo migration assay that traces adoptively transferred DC, IL-18 and IL-12 produced by neighboring keratinocytes and Langerhans cells regulate DETC OPN was injected in close proximity to draining LN at the time of intradermal DC transfer. function, and may play important roles in the regulation of immune responses in skin-associated Compared to controls OPN increased significantly the number of labelled DC attracted to the lymphoid tissues (SALT). regional LN. By RT-PCR, western-blot and ELISA we found that immature migratory DC strongly expressed OPN mRNA and secreted high levels of OPN. Of note, such OPN expression was downmodulated during DC maturation, associated with a loss of motility. These findings indicate that OPN is of importance for DC trafficking from peripheral tissues.

341 342 The Effects of Dexamethasone and Cyclosporine on the Activation of Dendritic Cells Induced Haptens Can Directly Induce Several Phenotypic and Functional Changes of Epidermal Langerhans by Various Stimuli Cells Required for Induction of Contact Dermatitis in Monocyte-derived Human Langerhans H. Manome, S. Affia, S. Singh* and H. Tagami Cells In Vitro Department of Dermatol, Tohoku University of School of Medicine, Sendai, Japan; *Department S. Affia, H. Manome and H. Tagami of Dermatol Banaras Hindu University, Varanasi, India Department of Dermatology, Tohoku University of School of Medicine, Sendai, Japan Immunosuppressive drugs such as corticosteroids and cyclosporine (CY) are used in the treatment We have reported that human monocyte-derived DCs (MoDC) obtained from the culture of of immune-mediated diseases. It has been already confirmed that they exert their immunomodula- peripheral blood monocytes (PBMC) with GM-CSF and IL-4 can be activated by a variety of tory action through their effects on T lymphocytes. On the other hands, their effects on antigen simple chemicals such as haptens and several metals. Recently, it has been demonstrated that TGF- presenting dendritic cells (DCs) are still controversial. Recent progress on DC biology has 01β1 can induce further differentiation of MoDCs to dendritic Langerhans cells with the expression demonstrated that DCs can be induced to increase their antigen presenting function via various of E-cadherin (Ecad), cutaneous leukocyte antigen (CLA), and Lag. In this study, using stimuli, e.g. cytokines, CD40 ligand, bacterial products, and haptens. In this study, to clarify the these cultured Langerhans cells, we examined whether representative haptens, i.e. NiCl2, difference in the efficacy of these two drugs on the activation of DCs by different stimuli, we dinitrochlorobenzene (DNCB) and dinitrofluorobenzene (DNFB), can induce their phenotypic examined their effects on CD86 and HLA-DR antigen expression and TNF-(x secretion by and functional changes as reported in murine or human Langerhans cells during sensitization phase monocyte-derived human DCs stimulated with two representative haptens, NiC12 and DNCB, of contact sensitivity reaction in vivo. The treatment with NiCl2 increased the expression of the lipoplysaccharide (LPS), superantigens, anti-CD40 antibody, or the combination of IL-1 PITNF- molecules related to antigen presentation, CD83, CD86, MHC class 1 and class 11, down- u. Dexamethasone (DEX) significantly suppressed the augmented expression of CD86 or HLA- regulated those required for homing to the skin and staying in the epidermis, CLA and E-cad, DR antigen by haptens, LPS, superantigens, anti-CD40 antibody, or IL-1 PITNF-(x at its and increased those essential for migration to the regional lymph nodes, CD49e, CD44 and its therapeutically effective concentration. CY suppressed the augmented expression of CD86 only variant 6. DNCB and DNFB also increased CD86 and down-regulated E-cad and CLA. They by haptens or superantigens. DEX suppressed them far more potently than CY. Finally, DEX and augmented allogeneic T cell stimulatory function by cultured Langerhans cells. These data suggest CY suppressed TNF-01α secretion similar to their inhibitory effects on CD86 augmentation. that cultured Langerhans cells derived from PBMC present a good in vitro model to study the These data clearly demonstrated the difference between the suppressive effects of DEX and CY biology of epidermal Langerhans cells, and that haptens directly induce phenotypic and functional on the activation of DCs. These differences may reflect their different therapeutic outcome. changes of epidermal Langerhans cells required for afferent phase of contact dermatitis. 580 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

343 344 Recombinant Cholera Toxin B Subunit Activates Murine Dendritic Cells In Vitro and In Vivo as Cutaneous B-cell Lymphoma: Comparison of Immunoglobulin Gene Analysis Using Southern an Adjuvant Blot and Gene Fingerprinting I. Isomura, A. Morita, Y. Yasuda,* M. Isaka* and K. Tochikubo,* T. Tsuji A. Woolford, E. Fraser-Andrews, R. Russell-Jones and S. Whittaker Department of Dermatology and *Microbiology, Nagoya City University Medical School, Skin Tumour Unit, St. John’s Institute Dermatology, London, UK Nagoya, Japan Immunoglobulin heavy chain (IgH) gene analysis in B-cell lymphoma was first performed using Cholera toxin (CT) is the most commonly used for mucosal adjuvant in experimental animals. Southern blot analysis (SBA), and more recently using polymerase chain reaction (PCR) based CT is composed of two subunits: a toxigenic A subunit (CTA), which activates the adenylate methods. We report our experience of IgH gene analysis, comparing results obtained using SBA cyclase, and a pentameric 11.6-kDa B subunit (CTB), which is responsible for CT binding to the and PCR fingerprinting analysis (FA). cell membrane GM1 gangliosides. To separate adjuvanticity from the toxic property (CTA), we Eighteen samples from patients with primary or secondary cutaneous B-cell lymphoma (CBCL), have purified recombinant CTB (rCTB) secreted by Bacillus brevis carrying pNU212-CTB on which SBA was performed were subjected to fingerprint analysis. SBA was performed using a (expression-secretion system). The purification step has been done with D-galactose-agarose standard technique with a JH probe. PCR fingerprinting was carried out on genomic DNA using absorption column (LPS free). The purified rCTB was well tolerated in nasal or intracutaneous consensus primers for each of the six VH families, and a consensus JH primer. PCR products administration. It has been successfully used for mucosal adjuvant to induce humoral immune were subjected to fingerprint analysis on a denaturing 6% polyacrylamide gel. responses. The purpose of this study was to determine whether rCTB has the capacity to activate A B-cell clone was detected using both techniques in 11 samples. A B-cell clone was detected cellular immunity, especially through dendritic cells (DC). To isolate splenic DC, we obtained only with FA in three cases, only with SBA in three cases, and by neither method in one case of splenocytes suspension from BALB/C mice, followed by collagease digestion, bovine serum primary CBCL. albumin gradient and plastic adherence. The purity of CD11cϩ DC was over 50%. We incubated In approximately two of three of cases the results of SBA and FA are concordant, indicating that DC with different concentrations of rCTB and analyzed the surface phenotypes with CD11cϩ FA is a useful diagnostic tool in CBCL. There is some evidence of improved sensitivity of FA, DC population. Induction of MHC class II (Ia) and B7-2 was observed after 48 h. Maximum and this PCR-based method is able to detect a clonal B-cell population in some cases where induction was observed at the concentration of 1 µg per ml. IL-12 secretion from DC was also histology is equivocal, or not available. However, somatic mutation in the primer region may upregulated by 1 µg per ml of rCTB. These effects were almost similar to those of 300 ng per account for the three cases where a clonal population was detected using SBA but not FA, if ml of LPS. The stimulated DC with rCTB exhibited higher capacity to activate naı¨ve allogeneic necessary where FA is negative, specific PCR primers for the leader sequences of VH regions T cells than unstimulated DC. Furthermore, we investigated in vivo effect to Langerhans cells by which are not subject to somatic mutation may improve clone detection. We would then advocate painting 1 µg per ml rCTB in aceton/olive oil on the shaved abdominal skin of BALB/C mice. proceeding to SBA for negative samples. Langerhans cells in the painted site showed fully mature features characterized by the extension of elongated and lamellar dendrites. Thus, these results show that rCTB is sufficient to stimulate DC and induce maturation in vivo as well as in vitro. These observations may lead to the development of effective and safe vaccines to induce cellular immune responses.

345 346 Photopheresis Induces Monocyte Differentiation into Functional Dendritic Antigen Presenting In Vitro Generated Langerhans Cells Integrate into an Organotypically Cultured Human Epidermal Cells Equivalent and Form Adherens Junctions with Keratinocytes (KC) C. Berger, A-L. Xu, D. Hanlon, C. Lee and R. Edelson M. Fremuth, E. Kriehuber, M. Kornar, K. Wolff and K. Rappersberger Department of Dermatology, Yale University, New Haven, Conneticut Department of Dermatology, University of Vienna, Wa¨hringer Gu¨rtel, Vienna, Austria In contrast to lymphocytes, which undergo apoptotic cell death after exposure to 8-methoxypsoralen Within the epidermis Langerhans cells express surface molecules that mediate a coordinate and ultraviolet A light (8MOPact), monocytes not only survive but become activated. Since interaction with the extracellular matrix and KC, e.g. CD44, E-cadherin [E-Cad]). Since a defined monocytes can differentiate into dendritic antigen presenting cells (DAPC), we explored whether morphomolecular structure between Langerhans cells and KC has not yet been observed we exposure to 8MOPact during extracorporeal photopheresis (ECP) may induce monocyte differenti- processed Langerhans cell equivalents that were generated from stem cells derived either from ation into DAPC, explaining in part the immunologic results of ECP. Therefore, we examined GM-CSF/TNF-01α/SCF-cord blood or adult peripheral blood in coculture with KC. A significant leukocytes from ECP-treated cutaneous T cell lymphoma (CTCL) patients for DAPC phenotypic fraction of Langerhans cell equivalents rapidly adhered to KC and by electron microscopy we and functional properties. detected organized adhesion structures that closely resembled adherens junctions. Investigation of Leukocytes were phenotyped with a panel of monoclonal antibodies detecting DAPC cell surface the molecular composition of these adherens structures in binding inhibition experiments showed antigens: CD83, X-11, as well as CD14, CD11c, class II, CD1a and CD80. Staining was analyzed that anti E-cad antibodies (abs) reduced Langerhans cell equivalents–KC interactions by 60%, by flow cytometric determination of the mean fluorescence intensity (MFI) and cytospin evaluation. whereas anti-P-cad-, anti-CD44-or anti CD45-blocking abs had no significant effect, indicating Functional studies included the capacity to stimulate allogeneic T cells in mixed leukocyte culture that Langerhans cell–KC adhesion is mediated by E-cad. In further experiments we investigated (MLC) and to ingest particles. the functionality of Langerhans cell equivalents associated E-cad expression: Langerhans cell In nine of 17 ECP-treated CTCL patients, monocytes episodically expressed significant levels of equivalents during their in vitro generation assembled in clusters, where they displayed a striking CD83 and/or X-11 and CD1a (~2-fold increase, p 01ഛ 0.01–p 01ഛ 0.001), in comparison to surface expression of E-cad and western blotting additionally revealed the presence of certain normal controls. Monocytes from untreated CTCL patients (N ϭ 9) did not express elevated catenins (aE-, P-catenin and pp120). The observations that (i) a major portion of E-cad and b- levels of DAPC markers. Monocytes obtained from ECP-treated CTCL patients exhibited further catenin was found in the detergent insoluble fraction and that E-cad coprecipitated with aE- and augmentation of DAPC markers, maximizing after 20 h incubation. Uptake of fluorescent b-catenin strenghtened our thougs that Langerhans cell-associated E-cad occurs in a catenin and molecules and latex beads indicated that the DAPC were actively phagocytic. The capacity to cytosceleton bound configuration and thus can mediate functional binding to KC. These stimulate in MLC was preserved or enhanced after overnight culture, indicating continued display experimental data were addflonally confirmed by the ultrastructural demonstration of adherens of histocompatibility molecules. junctions that formed beween Langerhans cells and in an organ culture. Taken together, our These data reveals that ECP induces substantial functional DAPC induction without requiring findings suggest that Langerhans cell–KC adhesion is mediated by E-cad via adherens junctions. addition of cytokines or leukocyte isolation/manipulation. Since overnight incubation of treated leukocytes maximizes differentiation of monocytes into DAPC, which may be responsible for the initiation of clinically relevant anti-CTCL immunologic responses, we suggest that modification of the ECP procedure merits examination. A phase I clinical trial in CTCL patients of ECP, incorporating over-night incubation of treated leukocytes to maximize DAPC maturation, is currently in progress.

347 348 Tonsillar B Cells Do Not Express the P Selectin Ligand-1 but a Significant Proportion Exhibits Hyaluronan Expressed on Keratinocytes Mediates Langerhans Cell Migration Effective E-and P-Selectin Ligand Activity M. Mohamadzadeh, M. Mummert, A. Takashima D. Armerding, R. C. Fuhlbrigge and J. D. Kieffer, T. S. Kupper Department of Dermatology, UT South-western Medical Center, Dallas, Texas Department of Dermatology, Harvard Institutes of Medicine, Boston, Massachusetts Previous studies in many laboratories have shown that CD44 expressed on Langerhans cells serves In contrast to T cells, monocytes, neutrophils, and dendritic cells, the nature of E and P selectin as an essential adhesion molecule for their migration. On the other hand, the substrate(s) to which ligands on B lymphocytes has not been carefully explored. In the present study, human tonsillar Langerhans cell-associated CD44 binds in the epidermis have remained unclear. We tested the B cells from children (n ϭ 16) were studied. These cells had the cell surface phenotype of resting, hypothesis that hyaluronan (HA), an extracellular matrix carbohydrate known to serve as a major IgMϩ/IgG– mature B cells. They did not express PSGL-1 by FACS analysis, using four different ligand of CD44, is expressed on keratinocytes (KC) and, thus, mediates Langerhans cell movement monoclonal and one polyclonal antibody preparation. Surprisingly, as many as 60% of the B cells in the epidermis. We observed that: (i) bovine proteoglycan, an experimental ligand of HA, binds in a given preparation did express cell surface HECA-452 reactive epitopes. When tested in a significantly to epidermal cells freshly isolated from BALB/C mouse skin, short-term cultured parallel plate flow chamber assay, tonsillar B cells bound effectively to E and P selectin and resisted KC, and the Pam 212 KC line; (ii) pretreatment of cells with hyaluronidase abrogates this binding shear stresses as high as 50 dyn/cm2. Enzymatic treatment of B cells revealed the following completely; and (iii) Pam 212 KC express mRNA for hyaluronate synthase and 01β-D- differences between the B cell E and P selectin ligands and the known T cell E and P selectin glucuronidase, the major enzymes required for HA synthesis. These observations indicate that KC ligands (CLA/PSGL-1). (i) E selectin ligand activity on B cells is sensitive to O-sialoglycoprotein produce and express significant amounts of HA on their surfaces, thus, validating the first part of endopeptidase (OSGE). (ii) P selectin ligand activity on B cells is OSGE and neuraminidase our hypothesis. To test the function of HA, we first employed the long-term Langerhans cell line resistant. For both of the above, the converse is true for T cells. Like PSGL-1 on T cells, the P XS106. FITC-conjugated HA showed significant binding to LPS-treated XS106 cells, but not to selectin ligand activity on B lymphocytes was sulfate dependant. Significant differences where also untreated cells, corroborating our observation that LPS treatment elevated surface expression of seen upon immunochemical analysis. Western blotting of B cell lysates with HECA-452 revealed CD44. Under physiological flow conditions, LPS-treated, but not untreated, XS106 cells showed an immunoreactive band 01ജ 240 kDa which, unlike PSGL-1 from other leukocytes, was marked rolling over HA-coated plates as well as over Pam 212 KC monolayers, and the rolling completely resistant to reducing conditions. Western blotting with PSGL-1 antibodies revealed was blocked Ͼ95% by preincubation of XS106 cells with HA in both systems. Addition of 250 no immunoreactive species. Thus, we conclude that the E and P selectin ligand(s) on tonsillar B µg per ml of HA in the standard skin organ cultures resulted in Ͼ80% inhibition of Langerhans cells are distinct from CLA/PSGL-1. Interestingly, both in vitro activated tonsillar B cells and cell migration into culture media, as measured by counting the IAϩ epidermal cells that remained peripheral blood B cells exhibited low levels of HECA-452 reactivity and minimal E and P in skin specimens and the IAϩ cells that were recovered from the media. Finally, we examined selectin activity. In contrast, these cells maintained high expression of 01α401β7 integrin and L the in vivo impact of injected HA on Langerhans cell migration triggered by topical application selectin. These data suggest that the role of the E and P selectin ligand(s) expressed in situ on of 0.5% DNFB onto ear skin. Langerhans cell migration, as assessed by counting the density of tonsillar B cells may have a role unrelated to trafficking and tissue homing. IAϩ epidermal LC, was blocked Ͼ95% by local injections of HA (30 µg01ϫ2/mouse) and Ͼ90% by i.v. injection of HA (500 µg01ϫ2/mouse). By contrast, injected chondroitin sulfate had no significant effect. Thus, we conclude that HA expressed on the surfaces of KC serves as a primary adhesion substrate for Langerhans cell migration through multilayered KC in epidermis. These observations form a conceptual basis for the development of new pharmacological agents that block Langerhans cell migration, an initial step for the induction of cellular immune responses against skin-relevant antigens. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 581

349 350 High Expression of Macrophage Migration Inhibitory Factor (MIF) in Human Melanoma Cells Apoptosis of Melanoma Cells and Lymphocytes in Primary and Metastatic Lesions of Human T. Shimizu, R. Abe, A. Ohkawara, J. Nishihira* Malignant Melanomas: Possible Involvement of Fas System Departments of Dermatology and *Central Research Institute, Hokkaido University School of T. Shukuwa and I. Katayama Medicine, Sapporo, Japan Department of Dermatology, Nagasaki University School of Medicine, Nagasaki, Japan Macrophage migration inhibitory factor (MIF) is known to function as a cytokine, hormone, and Recently, it has been reported that melanoma cells express Fas ligand (FasL), which would induce glucocorticoid-induced immunoregulator. We recently reported that the presence of MIF protein apoptosis of Fas-expressing lymphocytes. To elucidate the frequency of apoptosis of melanoma in the human epidermal keratinocytes, particularly in the proliferative basal cells. This finding cells and infiltrating lymphocytes in melanoma tissue, the specimens from primary and metastatic indicates that MIF may function to regulate cellular proliferation and differentiation in the lesions of the patients, divided into Group A (alive for longer than six years after surgery) (n ϭ epidermis in addition to its immunity and inflammation. In this study, we revealed for the first 5) and B (dead within four years) (n ϭ 5) obtained from 10 cases of human malignant melanomas time that human melanocytes and melanoma cells express MIF mRNA, and produce MIF protein. were analyzed by TUNEL staining. The TUNEL-positive rates of melanoma cells were 0.7%, Immunohistochemical analysis demonstrated that MIF was mostly localized in the cytoplasm of respectively, and there was no difference between primary and metastatic lesions. The TUNEL- the melanocytes and G361 cells, a widely available human melanoma cells. In particular, strong positive rates of lymphocytes were 4.7% in primary lesions and the rates were lower in metastatic positive staining was observed at the dendrites of these cells. Expression of MIF mRNA was lesion. The rates of Fas-positive cells in melanoma cell or lymphocyte population well correlated exceedingly higher in G361 cells than in normal cultured melanocytes. MIF content in the with the positive rates of TUNEL as well as FasL. The PCNA-positive rates of both melanoma supernatant of the cultured G361 cells were significantly higher than that of the melanocytes. The cells and lymphocytes in metastatic lesions were lower than those in primary ones. The positive result shows that the G361 cells produce more MIF protein and secrete extracellular space than rates of TUNEL, Fas and FasL in the melanoma cell and lymphocyte population from the patients the melanocytes. Taken together, these finding indicate that MIF may also participate in the of Group B were significantly lower than those of Group A, while the difference of PCNA- regulation of cell growth. positive rate between Group A and B was not significant. Therefore, it is possible that melanoma cells and infiltrating lymphocytes may undergo Fas-mediated apoptosis through the autocrine/ paracrine mechanism, and that the expression of Fas and FasL in melanoma cells and lymphocytes may affect the prognosis of melanoma patients, while the apoptosis of infiltrating lymphocytes in itself may not influence the prognosis of the patients (in corporation with T. Koji, Department of Histology and Cell Biology, Nagasaki University).

351 352 The Inhibitory Effect of 01α-Tocopheryl Ferulate on Melanogenesis Developmental Expression of c-kit, bcl-2, HMB-45 and DOPA Reaction in Human Fetal Skin Y. Funasaka and M. Ichihashi C. Chang and H. Yu Department of Dermatology, Kobe University School of Medicine, Kobe, Japan Department ofof Dermatology, Kaohsiung Medical College, Kaohsiung, Taiwan 01‰[capsigma]0001[captau]α-tocopheryl ferulate (01α-TF) is a compound of 01α-tocopherol Melanocytes originated from neural crest. They entered the epidermis at 7 wk EGA. To investigate and ferulic acid connected with ester bond, the latter is also an antioxidant, and could absorb the density, morphology, distribution and developmental waves of melanocytes both in human ultraviolet (UV) to keep tocopherol stable. We have already reported that 01α-TF inhibits melanin epidermis and dermis, we studied the fetal skin of 7–34 wk EGA by immunohistochemistry on production with inhibition of tyrosinase and DHICA polymerase activity in cultured human epidermal sheets or vertical sections using monoclonal antibodies c-kit, bcl-2, HMB-45 and melanocytes. In this study, the inhibitory effect of 01α-TF on UV-induced melanogenesis was DOPA staining. The phenotype of fetal melanocytes was consistently c-kitϩ/bcl-2ϩ/HMB-45ϩ, examined using brownish and white guinea pigs as well as human skin organ cultures for the but DOPA staining was different among epidermis, outer root sheath and matrix of hair follicle, purpose to study the cytokine profile and DNA damages involved in this system. 01α-TF and dermis. In epidermis DOPA-positive cells appeared first at 12–13 wk EGA in part of fetal significantly inhibited UVB-induced melanogenesis, on the other hand, -tocopherol or ferulic melanocyte. Both DOPA-positive and DOPA-negative cells migrated into hair follicle, mostly acid showed no such effect. Further, 01α-TF inhibited the formation of not CPD or 6–4 DOPA-negative cells resided in outer root sheath and DOPA-positive cells in matrix during hair photoproducts but 8OHdG, and attenuated UV-induced expression of proopiomelanocortin embryogenesis. They were fully dendritic and reached the peak density around 12–14 wk EGA, (POMC) gene. These results suggest that 01α-TF may be a good candidate for the litening agent and declined in density since 16 wk EGA. Another population of c-kitϩ/bcl-2ϩ/HMB-45ϩ/ which suppresses UV-induced melanogenesis with sparing DNA damages induced by oxidative DOPA– appeared in dermis since 15 wk EGA, which were bipolar in shape, cell density increased stress, and POMC gene expression. during 16–18 wk EGA and declined since 20 wk EGA. There were heterogeneous populations of melanocytes resided in human skin since fetal period. There were two waves of c-kitϩ/bcl- 2ϩ/HMB-45ϩ populations entry into human skin, the first one, in epidermis at 7 wk EGA and the second one, in dermis at 15 wk EGA. They were different in morphology, DOPA reaction, integration of hair formation and developmental wave.

353 354 Ultraviolet B-Radiation Induces Apoptosis of Melanocytes by Upregulation of p53 and bax, but DTIC Suppresses Heat Induced HSP72 (72-kDa Heat Shock Protein) in Melanoma Cell – Study Not by Downregulation of bcl-2 In vitro K. Park, Y. Kim and K. Kim M. Hatoko, A. Tanaka, H. Tada, A. Tsubakimoto and T. Muramatsu Department of Dermatology, Seoul National University, Seoul, Korea; and Department of Division of Plastic Surgery, Dermatology, Nara Medical University, Kashihara, Japan Dermatology, Effiji Medical College, Taejeon, Korea Hyperthermia is one of the least invasive therapies for malignant tumor. However, hyperthermia DNA-damaging agents such as ionizing radiation (IR) and ultraviolet (UV) radiation activate the alone is often ineffective, and the combination therapy of hyperthermia and anticancer drugs have tumor suppressor p53 and in some cases cause apoptosis. Melanocytes, originated from embryonal been used to enhance thermosensitivity. Various hypothetical mechanisms of this thermoenhance- neural crest, make melanin granules to protect harmful UV lights from penetrating through the ment of anticancer drug have been proposed. Of these, some investigaters suggest that 72-kDa skin. It is reported that the p53 tumor suppressor gene product can induce apoptotic cell death heat shock protein (HSP72) has close relation to thermoenhancement of anticancer drug. through regulation of Bcl-2 and Bax gene expression. Melanocytes express high level of Bcl-2 In the present study, we investigated the influence of DTIC on heat induced HSP72 in melanoma and their susceptibility to apoptotic stimuli such as UVB is controversial. In this work, we tried cell. HMV-1 melanoma cell line were incubated at 37 in 25 cm2 flask, and the cells were heated to demonstrate evidence of apoptosis in UVB irradiated melanocytes and changes of Bcl-2 and at 44 for 30 min with various concentrations of DTIC. In all concentrations of DTIC studied, Bax after UVB irradiation. Normal human melanocytes were grown and exposed to UVB (0– the cell survival rate after combined treatment decreased more than those when two treatments 100 mJ per cm2) irradiation. We show that UVB irradiation with serum deprivation induces were done separately. Moreover, the level of induced HSP72 after combined treatment were apoptotic death of melanocytes. UVB irradiation also increases expression of p53 and Bax in dose- lower than those after heat treatment alone. These results suggest that DTIC, which enhances dependent manner, but does not affect the level of Bcl-2 in melanocytes. heat sensitivity of melanoma cell, suppresses heat induced HSP72. 582 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

355 356 Difference of p21 and p53 Induction After Hyperthermic Treatment Among Malignant Tumor Caveolin-1 is Decreased in Metastatic Human Melanoma Cell Lines in contrast to Flotillin-2 Cell – Study In Vitro P. Hazarika, M. R. Van Arsdall, R. McMillen, M. Bar-Eli and M. Duvic A. Tanka, M. Hatoko, H. Tada, A. Tsubakimoto and T. Muramatsu Section of Dermatology and Department of Cell Biology, MD Anderson Cancer Center, Division of Plastic Surgery, Dermatology, Nara Medical University, Kashihara, Nara, Japan Houston, Texas It is well known that the effect of hyperthermic treatment varies among tumor cells.On the other Caveolae are specialized, lipid rich, invaginated microdomains of the inner plasma membrane that hand, p53 and p21 are important proteins to analyze cellular responces against not only DNA facilitate vesicular trafficking and signal transduction by sequestration of growth factor receptors. damaging agents but also physiological stress such as heat. In the present study, we investigate the Caveolin-1 (Cav-1), a 21-kDa protein, is a predominant component of caveolae, and has been induction of p21 and p53 to know the variation of cellular responce between tumor cells including implicated in cell proliferation, transformation, and negative regulation of signal transduction. melanoma and SCC after hyperthermic treatment in vitro. Flotillin-2 (Flot-2), is a 42-kDa caveolae-associated protein. Flot-2 expression is associated with Two kinds of human cutaneous malignant melanoma cell lines (HMV-1, HM6KO), SCC cell filopodia formation in cos-1 epithelial cells. To determine the relevance of caveolae associated line (A431) and glioblastoma cell line (A172) were used in this study. Heat treatment was proteins Cav-1 and Flot-2 to oncogenesis and metastasis, we studied the expression of these performed at 44 for 30 min. The survival rate of cells after treatment was determined using the proteins in six human melanoma cell lines by immunofluorescence (IF) and by western blotting. clonogenic assay. Survival rate was 0.69 in HM6KO, 0.61 in HMV-1, 0.17 in A172 and 0.14 in The nonmetastatic line (SB2) strongly stained with anticav-1 but not 2 highly invasive metastatic A431.Western blot analysis showed that in A172, both of p21 and p53 were induced, while in lines (MeWo & TXM 13). By immunoblotting, flot-2 protein was expressed at a constant level HMV-1, only p21 was induced. In HM6KO and A431, only p53 was induced. in all lines, suggesting that it may serve a critical function for the cells. In contrast, Cav-1 was Four cell lines variy in the survival rate and the induction of p53 and p21 proteins. These results differentially expressed across the melanoma lines. Highly metastatic cell lines had decreased may suggested that the difference of the effect of hyperthermic treatment has a relation with the expression of Cav-1 compared to nonmetastatic cells. Loss of Cav-1 expression appears to be a difference of the induction of p53 and p21 proteins. significant factor in melanoma progression and may function in cell signaling.

357 358 An Improved Quantitative Assay for Melanin Production and Release by Cultured Mouse and TFE3 and TFEB as Transcriptional Activators of Tyrosinase and TRP1 Genes Human Melanocytes after Testosterone and UVA Induction C. Verastegui, C. Bertolotto, K. Bille, P. Abbe, J. P. Ortonne and R. Ballotti J. Yang, R. Krajcik, C. O’Bryan and N. Orentreich INSERM U385 Nice, France Orentreich Foundation for the Advancement of Science, Inc., Cold Spring, New York Microphthalmia, a basic helix-loop-helix-leucine zipper (bHLH-Zip) transcription factor involved Skin melanin serves not only a cosmetic purpose but also provides protection against ultraviolet in the development of the melanocyte lineage has been shown to play a key role in the light. Although skin color is related to the production of melanin by melanocytes, it is transcriptional regulation of the melanogenic enzymes, tyrosinase and TRP1. Noteworthy, among also dependent upon the distribution of melanin through melanocytic dendrites to epidermal the transcription factors of the bHLH-Zip family, TFE3 and TFEB show a remarkably elevated keratinocytes. Therefore, any assay used to evaluate compounds for their effects on pigment homology with microphthalmia, particularly in the bHLH and in the N-tenninal transactivation production must measure both intracellular and extracellular melanin. In this study, an in vitro domains. These observations prompted us to investigate the role of TFE3 and TFEB in the quantitative assay for intra- and extra-cellular melanin using small numbers of mouse B16 and regulation of tyrosinase and TRP1 gene transcription. Here we show that TFE3 and TFEB are human HTB cells was developed. Five 01ϫ 104 B16 cells per well in DMEM or 2 01ϫ 105 expressed in melanocyte. As previously reported for microphthalmia, TFE3 binds to M box HTB cells in MEM were incubated for 5–7 d in the presence or absence of a test compound (AGTCATGTGCT) and an E box motifs (CATGTG) surrounding the TATA box and stimulates with and without UVA exposure. The cells were collected and counted in a hemocytometer both the tyrosinase gene transcription. Further, TFE3 and TFEB, through their binding to the M box for total cell number (cell proliferation index) and number of pigmented cells versus unpigmented motif, stimulate the TRP1 promoter activity. These data indicate that TFE3 and TFEB are able (pigmentation index). The cells were then solubilized in 1N NaOH, and melanin was measured to regulate tyrosinase and TRP1 expression, making of these transcription factors potential actors spectrophotometrically at 475 nm. The data was normalized to cell number. The supernatant was in the regulation of melanogenesis and in the differentiation of pigmented cells. also collected and centrifuged; the pellet, which contains secreted melanin, was suspended in 1N NaOH and read at 475 nm. In vitro, mouse melanocytes accumulate pigment and release very little; in contrast, human cells release their melanin granules and never appear as dark as the mouse cells. Both testosterone (T) and epitestosterone (Epi-T) increased melanin production up to 2.5- fold in a dose dependent manner. Human melanocyte nucleii stain positive for the androgen receptor (AR), and the AR blocker ﬂutamide completely inhibited the effects of T and Epi-T on melanogenesis. Combination treatment of UVA with either T or Epi-T had a synergistic effect on both melanogenesis and melanin release, a phenomenon which may relate to tanning of human skin.

359 360 UVB-Control of Melanogenesis via Regulation of CD10/E.24.11, the Enzyme Responsible for Nitric Oxide Synthase Expression and Apoptosis in Psoriasis the Degradation of 01αMSH S. Chang, S. Jung and T. Yoon E. Aberdam, B. Mari,* P. Auberger,* J-P. Ortonne and R. Ballotti Department of Dermatology, Chungbuk National University, Cheongju, Korea INSERM U385 and CJF 96–05,* Nice, France Nitric Oxide (NO) has been found to be important in a number of different physiological Exposure of mammalian skin to ultraviolet radiation increases melanin synthesis by cutaneous processes. Of particular relevance to the skin are the roles of NO in vasodilatation, inflammation, melanocytes which protects against photodamage and photocarcinogenesis. immunomodulation and in oxidative damage to cells and tissues. NO exhibits contradictory effects Several observations have suggested a molecular basis for UVR-induced melanogenesis that in the regulation of apoptosis. Psoriasis is a common chronic skin disease mediated by cellular includes activation of the MSH/MSH receptor system as a control event. However, very few is immune mechanisms. Recently, apoptosis is considered as psoriasis pathophysiology by many known about the degradation of this peptide leading to the end of the melanogenic signal. In this investigators. The purpose of this study was to investigate the role of NO in psoriasis pathogenesis, study, we have focused our attention on a metalloprotease, the endopeptidase 24.11 or CD10 such as inflammation, dermal vessels dilatation, apoptosis. Immunohistochemical stainings with which has been previously described to cleave 01αMSH in a cell free system. We have shown monoclonal antibody to iNOS and polyclonal antibodies to eNOS, PCNA, bcl-2 and p53, were that this enzyme is expressed at the surface of human melanocytes and that its activity is down- performed on paraffin-embedded tissues. TUNEL stainings were performed for apoptotic cells. regulated by UVB treatment reaching 80% inhibition as compared to basal levels (ranging between Western blot with monoclonal antibody to iNOS and polyclonal antibodies to eNOS were 1.5 and 8 nmoles per mg.min depending on the cultures tested). Interestingly, we observed a 3- performed on psoriatic and normal skin tissues. The results were as follows: western blot with fold activation of E.24.11 activity when the melanocytes were treated with 01αTNF, a potent iNOS showed 130 kDa positive band in psoriasis, but not in normal skin tissues. Western blot inhibitor of melanogenesis. Moreover, we observed a significant left-shift of the 01αMSH dose with eNOS showed 135 kDa band in all psoriatic and normal skin tissues. Immunohistochemical dependent dopa -oxydase reaction in the presence of phosphoramidon, a specific inhibitor of stainings with iNOS, eNOS showed positive reactions in psoriasis. But in eNOS staining, epidermis E.24.11. This result confirms the biological relevance of our observations. Taken together, these tended to be weaker stained than dermal vessels. TUNEL staining showed increased apoptosis in data shed light to a new and interesting way of UVB-induced regulation of melanogenesis via psoriasis. The results suggested that NO was considered to play a role in psoriasis pathogenesis, control of 01αMSH degradation. including apoptosis, dermal vessels dilatation and inflammatory infiltration. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 583

361 362 Promoted Dermal Fibrosis During Wound Healing in Calponin h 1 (CNhl)-Deleted Mice Expression of the Cyclin-Dependent Kinase Inhibitor p27(kip) in Keratoacanthoma M. Takeoka, T. Ehara, H. Murata, T. Saida, M. Katsuki, K. Takahashi and S. Taniguchi W. Hu, T. Cook, C. Oh and N. Penneys Department of Molecular Oncology and. Angiology, Department of Pathology, Department of Department of Dermatology, St. Louis University, St. Louis, Missouri; Department of Dermatology, Dermatology, Shinshu University School of Medicine, Matsumoto; Institute of Medical Science, Gyeong-Sang National University, Chinju, South Korea Tokyo University, Tokyo; Osaka Medical Center for Cancer and Cardiovascular Disease, Keratoacanthomas are characterized by initial rapid enlargement followed by clinical regression. Osaka, Japan p27(kip), an inhibitor of cyclin-cyclin-dependent kinase complexes, may act to hold eukaryotic CNh1 is a calmodulin- and actin-binding protein expressed in smooth muscle cells and fibroblasts. cells in a quiescent state (G0). We examined p27(kip) expression in expanding (EKA) and regressing It promotes actin polymerization and inhibits actin-activated myosin ATPase activity, and is known (RKA) keratoacanthomas with an immunohistochemical method. The avidin-biotin-peroxidase to regulate cell adhesion, proliferation as well as cell motility through the interaction with actin complex system was used to demonstrate p27(kip) in 6 µm sections from five cases of EKA and filament system in vitro. To investigate the functions in vivo, we generated CNhl deleted mice by 15 cases of RKA. EKA was a crateriform tumor of atypical epithelium characterized by minimal replacing pMC1neopGKgpt with the exon 5–7 of CNhl -gene. inflammation and tumor-associated stroma; RKA had similar epidermal changes associated with Severer dermal fibrosis was observed in the process of wound heating in abdominal skin and tail inflammation and tumor-associated stroma. p27(kip)-labeled cells were counted for each case in end in the calponin-deleted mice than in the wild mice. More fibroblasts were stained in the 4 high power fields of tumor epithelium that contained the most obvious labeling. wound region by immunostaining with anti-PCNA. Furthermore, the proliferation of fibroblasts P27(kip) was found overlying nuclei of suprabasilar keratinocytes in normal epidermis. In EKA, cultured from the skin of CNh1-deleted mice was enhanced as compared to that of wild mice. It there was rare expression of p27(kip) overlying nuclei in the neoplasm (1.25 Ϯ 2.1 labeled cells is suggested that CNhl plays a role to suppress the overgrowth of fibroblasts, and that this CNhl- per high power field); in RKA, there is dramatic expression of p27(kip) overlying nuclei in the deleted mouse could be a model for keloidosis. neoplasm (55.1 Ϯ 28.6 labeled cells per high power field). The difference was significant at p Ͻ 0.001 using the Mann–Whitney Rank Sum test. Expression of p27(kip) in keratoacanthoma may be a controlling mechanism for regression by inhibition of progression into S phase.

363 364 The Effect of the FGF-5 Protein and the FGF-5S Protein on the Mouse Hair Growth Cycle Localization of Transglutaminase Activity in Developing Human Hair Follicles S. Suzuki, Y. Ota, K. Ozawa* and T. Imamura* M. Akiyama, L. T. Smith* and H. Shimizu† POLA R&D Laboratories, Yokohama; and *Biosignaling Department, National Institute of Division of Dermatology, Kitasato Institute Hospital, Tokyo, Japan; *Departments of Biological Bioscience and Human Technology, Tsukuba, Japan Structure and Medicine/Dermatology, University of Washington School of Medicine, Seattle, The Fgf-5 gene produces a short form of the FGF-5 protein (FGF-5S) as well as the full-length Washington; †Department of Dermatology, Keio University School of Medicine, Tokyo, Japan FGF-5 protein (Ozawa K et al. Mol Brain Res 41:279–288, 1996). We previously reported that Epidermal cornified cell envelope (CCE) formation is an important step toward the final stage of the murine FGF-5 protein and the FGF-5S protein locate in the macrophage-like round cells of keratinization. CCE precursor proteins including involucrin, loricrin and small proline-rich proteins the skin and the cortex of the hair follicles, respectively, and that appearance of these two products 1/2 are cross-linked by keratinocyte transglutaminase (TGase), mainly TGase 1, to the inner are associated with the hair growth cycle (Suzuki S et al. J Invest Dermatol, 1998, in press). In the surface of plasma membrane of cornifying cells. CCE outer surface is coated with material derived present study, we observed the effect of FGF-5 and FGF-5S on the mouse hair growth cycle. By from secreted lamellar granules. In order to clarify the time and localization of TGase activity in depilation, anagen was induced in the back skin of mice in the telogen phase of the hair cycle. developing human hair follicles, skin samples from human fetuses of a series of estimated gestational Mice were administered a dorsal subcutaneous injection of FGF-5 or FGF-5S (1 or 5 mtg per ages (EGAs) were examined using in situ assays of TGase activity by dansyl cadaverine incorporation. body) once daily for 7 d from the beginning of the anagen. Only mice which had received FGF- Inner root sheath cells of the bulbous hair peg (105–135 d EGA) showed strong TGase activity. 5 injection showed the delay of the hair growth at the injection site, suggesting that FGF-5 Subsequently, TGase activity was detected in primitive hair canals and cuticle of the late bulbous inhibits the hair growth in the anagen I–V. Because it was demonstrated that the functions of hair peg (120–135 d EGA) and in those of lanugo hair follicle (Ͼ135 d EGA). Immunohistologically, FGF-5 are partially antagonized by FGF-5S (Ozawa K et al. J Biol Chem 273:29262–29271, 1998), the similar ontogenic expression patterns were observed in major CCE-related molecules including the inhibition of the hair growth by FGF-5 may also be antagonized by FGF-5S. We are now TGase 1/3, involucrin, loricrin, small proline-rich proteins 1/2 and a 25-kDa lamellar granule- observing the effects of FGF-5 and FGF-5S on the catagen induction. associated protein. These data indicated that TGase activity is expressed in sites defining the hair canal and cuticle formation in accordance with the expression of other major CCE-related proteins during human hair follicle development.

365 366 Elevated RANTES in Plasma or Skin and Decreased Plasma IL-10 in Subsets of Patients with Expression of Desmoglein and Plakoglobin in Skin Carcinomas Severe Atopic Dermatitis H. Tada, A. Tanaka, A. Tsubakimoto, M. Hatoko and T. Muramatsu Y. Niwa Division Of Plastic Surgery, Dermatology, Nara Medical University, Kashihara, Nara, Japan Niwa Institute for Immunology, Tosashimizu, Japan Reduction or absence of cell adhesion molecules has been reported in various carinomas and the The 01β-chemokine RANTES was measured in not serum but plasma, and in skin of 197 patients abnormal expression of these molecules contributes to the invasive and metastatic behaviour of with atopic dermatitis (AD). Plasma levels were also determined in all patients for the platelet- malignant tumor cells. derived chemokine 01β-thrombglobulin (01β-TG) and for IFN-01γ, IL-2, IL-4, IL-5, and IL- In epidermal keratinocytes, main cell adhesion systems are adherens junctions and desmosomes. 10, along with serum IgE levels and blood eosinophils. One hundred eight of the patients had In the present study, we examined the expression of desmoglein and plakoglobin, the constitutional severe AD. Of these, one third showed a marked increase in plasma RANTES and a decrease in components of desmosomes called desmosomal cadherin, of various skin carcinomas such as basal plasma IL-10 levels, neither of which was seen in the remainder of the patients. In in vitro cell carcinoma (BCC), squamous cell carcinoma (SCC) and Paget’s disease by immunoﬂuorescence experiment, IL-10 production by stimulated lymphocytes was also lowered in the presence of method. In normal human skin, desmoglein and plakoglobin were strongly expressed in the high concentration of RANTES. Platelet RANTES content correlated inversely with plasma intercellular space of the epidermis. In BCC and SCC, the expression of these molecules was RANTES level, and e-TG showed a pattern qualitatively similar to that of RANTES, both in markedly reduced or absent in tumor cells. In carcinoma in situ of Paget’s disease, compared with plasma and in platelets. Plasma levels of IFN-01γ, IL-2, and IL-5, serum IgE and blood eosinophils the normal epidermal cells surrounding tumor cell nests, the expression of these molecules was all were elevated in direct relation to the severity of the AD. In skin biopsy, the 32 patients with reduced in tumor cells. In the invasive type of Paget’s disease, the expression of these molecules the most severe disease had a higher RANTES content in both lesional and healing skin than the was almost absent throughout the epidermis. These results suggest that the expression of desmosomal other biopsied patients. Our data suggest that locally produced RANTES may play a critical role cadherin is reduced or absent in human skin carcinomas, and that reduction of these molecules in the persistence and recurrence of AD, whereas high circulating levels of RANTES, perhaps may also contribute to the invasiveness and metastasis of skin carcinomas. released from platelets by IgE-mediated stimulation, may promote AD both directly and through an inﬂuence on the Th1/Th2 cytokine balance. 584 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

367 368 Retinoid – Inducible Class II Tumor Suppressor Gene (TIG-3) is Significantly Decreased in Role of T-cells Bearing Receptors (CD94, CD158, CD161) for MHC Class I and Nonclassical Squamous Carcinomas MHC-like (CD1d) antigens in psoriasis B. Helekar, D. DiMao, D. Lipton, J. Breuer-McHam, D. DiSepio, S. Nagpal, G. Clayman, S. B. J. Nickoloff, B. K. Bonish and S. A. Porcelli Strom, M. Kripke, R. Chandraratna and M. Duvic Skin Disease Research Laboratories, Departments of Pathology and Medicine, Loyola University, Department of Medical Specialties – Section of Dermatology, Departments of Immunology, Head Maywood, Illinois; Brigham and Women’s Hospital, Boston, Massachusetts and Neck Surgery, MD Anderson Cancer Center, Houston, Texas; and Allergan Research, The precise immunologic and genetic basis for psoriasis is uncertain. T-cells expressing C-type Irvine, California lectins (CD94, CD161), and other receptors present on natural killer cells (NKRs) (CD158a, Tazarotene inducible gene 3 (TIG-3) was isolated by differential display PCR from retinoid CD158b, NKB1) may correspond to NK-T cells that become activated by interaction with treated skin equivalents and has 52% homology to H-rev 107, a class II tumor suppressor. Tig-3 specific HLA alleles or CD1d. A previously overlooked population of immunocytes was detected causes growth inhibition in vitro and is inducible in breast carcinoma lines that are inhibited by by immunostaining skin samples (n ϭ 6) using commercially available mAbs. Only rare to absent retinoic acid (in press, PNAS, 1999). To determine the possible role of TIG3 in squamous NK T-cells in epidermis of normal healthy skin or prepsoriatic skin, Bowen’s disease, basal cell oncogenesis and epithelial differentiation, in situ hybridization was performed to localize mRNA carcinoma or squamous cell carcinoma was present. However, while peripheral blood contains expression in tumors and paired normal specimens including 13 aggressive cutaneous squamous only 1% NK T-cells, approximately 5%–10% of intraepidermal lymphocytes in psoriatic plaques carcinomas (SSC), nine nonaggressive cutaneous squamous carcinomas, three basal cell carcinomas, (n ϭ 9) were NK T-cells with a relative predominance of CD161 Ͼ CD94 Ͼ CD158b Ͼ NKB1 two breast carcinomas, four lung cancers and one ovarian carcinoma. Digoxigenin antibody was Ͼ CD158a. 2 color labeling revealed that CD94 predominated in CD8ϩ versus CD4ϩ cells. used to detect hybridization of UTP-labeled sense and antisense RNA probes, and two blinded Moreover, as CD1d was believed to be restricted to intestinal epithelium, we discovered CD1d observers used a semiquantitative grading scale from 0 to 5 to score the degree of staining. Non- is also expressed in skin (i.e. keratinocytes, KCs; pilosebaceous unit; dendritic cells; endothelium), parametric comparisons were performed using the Wilcoxon test for paired and Mann–Whitney and is markedly enhanced (compared to the diseases above) in psoriatic epidermis with activated test for nonpaired data. TIG-3 mRNA is highly expressed in the suprabasal layers of normal appearing CD161 ϩ NK T-cells extending cytoplasmic spikes around KCs with prominent surface epidermis and in adenexa. Basal epidermis and basal carcinomas have 2-fold less than suprabasal, CD1d. In the SCID-Hu xenograft model, injection of activated allogeneic immunocytes into differentiated layers. There was significantly less TIG-3 in all squamous cell carcinomas compared normal skin unexpectedly produced marked epidermal hyperplasia in both human and adjacent to overlying normal skin (p ϭ 0.0001), as well as to adjacent normal skin (p ϭ 0.007). Aggressive mouse skin. The evolutionarily conserved in its function, CD1d, was expressed by both mouse SCCs had significantly less TIG3 expression than nonaggressive SCC (p ϭ 0.038) and all aggressive and human KCs which were infiltrated by human CD161 ϩ NK T-cells. Another functional link SCC studied showed decreased TIG-3. Bronchoalveolar lung carcinoma had complete loss of was observed in a separate set of grafts in which pretreating immunocytes with blocking mAbs TIG-3. Partial loss was detected in lung carcinoid and squamous cell carcinomas and in endometrial against NKRs prevented induction of psoriasis. Thus, T-cells bearing NKRs for both classical carcinoma of ovary. TIG3 was expressed only in ductal epithelium of normal breast and was MHC antigens as well as CD1d, are present in lesions which represent novel immunointervention heterogeneous in breast carcinomas. Low TIG-3 expression appears to be associated with the targets for psoriasis. proliferative normal basal cell layers and squamous cell carcinomas and upregulation of TIG-3 expression may influence normal epidermal differentiation.

369 370 The Seattle Melanoma-Dysplastic Nevus Project: A Case-Control Analysis Autolmmune Alopecia in New Zealand Black/KN Mouse is Regulated by Y Chromosome S. Kim and M. Piepkorn Associated Antigens but not by H-2 Complex or Immune Complexes Deposits University of Washington School of Medicine, Seattle, Washington F. Furukawa, T. Ito, N. Sco, Y. Tokura, M. Takigawa and M. Ito The clinical significance of dysplastic nevi and their relationship to melanoma continue to be Department of Dermatology, Harnamatsu University School of Medicine; Department of Dermato- debated. We have implemented a case-control study wherein newly incident cases of melanoma logy, School of Medicine, Nfigata University, Japan are enrolled along with their spouses as controls. The novel aspect of the design is that the largest, Autoimmune alopecia is one of the major symptoms in systemic lupus erythematosus (SLE). most clinically atypical macular nevus from each case and control subject is biopsied for histological Although the in vivo immune complexes deposits and mononuclear cell infiltrates have been analysis of dysplasia. The numbers of nevi Ͼ 2 mm on the backs and left arm are counted, and proposed as the cause, the mechanisms are still obscure. A male New Zealand Black (NZB)/KN the participants complete a questionnaire regarding sun exposure and demographic variables. An mouse is a novel SLE-prone mouse associated with arthritis, which is characteristic in spontaneous interim analysis (n ϭ 75 subjects) has shown significant differences between cases and controls alopecia lesions. We designed the genetic studies on autoinimune alopecia in NZB/KN mice in with respect to nevus counts. The mean counts on the left arm of cases and controls are 10.2 and relation to several autoimmune traits. 6.9, respectively (p ϭ 0.07) and on the back are 18.8 and 12.0 (p ϭ 0.05). This trend is concordant NZB/KN(H-2d), the standard SLE-prone NZB/N(H-2d), the control NZ While (NZW)/sk (H- with results from previous case-control studies. Mean numbers of nevi Ͼ 5 mm are not significantly 2z) mice were purchased and bred in our laboratory. F1 hybrid mice of (NZW/sk 01ϫ NZB/ different for cases and controls. Single observer analysis of the biopsy slides showed the architectural KN) (H-2z/d) and 92 F2 mice of (F1 01ϫ F1) (H-2d/d, H-2z/d, H-2z/z) were raised. In F2 mice, pattern of dysplasia with mild melanocytic atypia to be present in Ͼ 50% of nevi from both case the correlation studies were performed. and control subjects, indicating that these features are nonspecific. Three instances of more Male NZB/KN mice showed alopecia lesions without erythema on the upper back and the tail advanced melanocytic atypia, however, were observed, and each of these nevi had been removed regions in 25% and 80% at the age of 3 mo and 12 mo. The early alopecia lesions showed a slight from melanoma cases, suggesting a weak association between melanocytic atypia and predisposition number of infiltrates around hair follicles. No alopecia lesions were found in NZB/N or NZW/ to melanoma. This trend will be further evaluated with the accrual of additional subjects, but this sk mice. Male and female F1 mice showed alopecia in 13.3% and 6.7%. F2 developed alopecia in interim analysis suggests that our study population will be adequately informative to evaluate select 44% of male mice and 7.3% of female mice. Strong positive association was found between aspects of the epidemiological relationship between melanoma and dysplastic nevi. alopecia andmale, but not among alopecia, female and H-2 complex. IgM and IgG deposits at hair follicle basement membrane were found in 75% and 5% of 12 mo-old NZB/KN mice, and in zero of male NZB/N mice or NZW/sk mice. Male F2 mice showed 53% positivity of IgM deposits and 33% of IgG deposits, and female F2 showed similar incidences of Ig deposits. No associations were found among alopecia, Ig deposits, H-2 complex and proteinuria. These results suggest that autoiramune alopecia in NZB/KN mice are closely associated with Y chromosome associated antigens (Yaa), but not with H-2 complex or in vivo Ig deposits. Since lupus dermatoses in other SLE-prone mice such as NZB/N and MRL/lpr mice are regulated by H-2 complex or lpr gene, it is likely that the pathogenesis of autoinimune alopecia is different from that of erythema and the related immune complex deposits.

371 372 Immunofluorescent Microscopic Investigation of the Distal Arrector Pili: A Demonstration of the Expression of Laminin 01α1, 01α2, 01α3, 01α4 Chains During Skin Morphogenesis Requires Spatial Relationship Between Alpha-5 and Fibronectin an Epithelial–Mesenchymal Interaction M. Clifton,*† T. D. Horn,*† B. R. Smoller,*† J. Mendelson,* D. Montague,‡ and C. Carter§ R. Fleischmajer, A. Utani, E. McDonald, A. Sapadin and Y. Yamada Departments of *Dermatology, †Pathology, ‡Orthopedics, and §Gynecology, University of Department of Dermatology, Mount Sinai Medical Center, New York, New York and National Arkansas for Medical Sciences, Little Rock, Arkansas Institute of Dental Research, Bethesda, Maryland Our previous studies of the arrector pili (AP) have shown that the 01α501β1 integrin subunit is The purpose of this study was to determine mRNA expression of laminin (Ln) 01α1, 01α2, found on the distal AP fibers. Putative ligands for 01α501β1 include collagen type I and 01α3, 01α4 chains in human keratinocytes and dermal fibroblasts monocultures and following fibronectin. The purpose of this study is to create an immunoflourescent three-dimensional their recombination into a 3-dimensional skin equivalent. After 4 wk in culture, the epidermis (3D) picture of the spatial relationship between 01α5 and fibronectin at the AP-extracellular was separated from the dermis by thermolysin digestion; total RNA was extracted; primers matrix junction. prepared for Ln 01α1, 01α2, 01α3, 01α4 chains and Ln 01γ1, 01γ2 chains; mRNA estimated by Serial 5 micron frozen sections of human scalp skin were double-labeled via immunoflourescent RT-PCR. Fibroblasts in monocultures did not express any Ln 01α chain. However, after coculture staining for 01α5 with flourescein and fibronectin with rhodamine. Flourescent microscopy was with keratinocytes, there was strong expression of Ln 01α1, 01α2, 01α3, 01α4 mRNAs. used to visualize and photograph the images for computerized 3D reconstruction. Keratinocyte monocultures only expressed the Ln 01α3 chain but after coculture with fibroblasts Flourescent microscopy showed the typical granular staining of the AP muscle fibers with they also expressed the Ln 01α1, 01α2 chains, but not the Ln 01α4 chain. Quantitative RT-PCR flourescein and smooth staining with rhodamine. Overlapping staining of 01α5 and fibronectin of the Ln 01γ1 chain (present in all laminins except for Ln-5) showed that 80% derives from the on the peripheral fibers of the AP muscle was seen using a dual filter. A 3D reconstruciton further dermis and 20% from the epidermis. The Ln 01γ2 chain mostly derived from the epidermis. demonstrates the colocalization of these epitopes. Immunocytochemistry with mono-clonal antibodies localized the Ln 01α1 and 01α3 chains in These results support our hypothesis that 01α5 and fibronectin mediate the attachment of the keratinocytes while the Ln 01α2 chain was expressed in the dermis. The Ln 01γ1 chain was noted distal AP to the extracellular matrix. at the epidermal–dermal interface, while the Ln 01γ2 chain was restricted to the hemidesmosomes. This study showed that although most laminins derive from the dermis, the expression of Ln 01γ chains requires a bi-directional epithelial–mesenchymal interaction. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 585

373 374 A Role of Integrins to Maintain Thickness and Physical Properties of Hairless Rat Skin Stimulation of Keratinocyte Type IV Collagen Synthesis by Soya Sapogenols S. Moriwaki, T. Fujimura, K. Tsukahara, T. Kitahara and Y. Takema F. Bonte´, C. Gondran, P. Barre´ and M. Dumas Kao Biological Science Laboratory, Tochigi, Japan LVMH Laboratory of R&D, Branche Parfums Cosme´tiques, Saint Jean de Braye, France The extract of Fucus vesiculosus accelerates contraction of collagen gel by fibroblasts due to an The mecanical stability of the basement membrane at the dermis–epidermis interface is mainly increase in integrin 01α2 and 01β1 molecules. An intradermal injection of 5 µl of anti rat 01α2 due to anchoring plaques formed by type IV collagen. A lack of type IV collagen is believed to and 01β1 antibody mixture (0.75 mg per ml each) into rat dorsal skin resulted in a significant participate to wrinkles formation. We have used an antihuman type IV collagen antibody and increase in skin thickness for more than 3 h. The decrease in physical values of skin (Ue and Uf) immunogold followed by image analysis to measure a decrease of type IV collagen in the lamina was similar to the changes of those values in skin edema case. The topical application of the Fucus densa and at the anchoring plaques of aged and wrinkled skin biopsies. We then determined extract (0.3w/w%, 0.5 mL) on the dorsal skin of rats for 3 wk induced a significant decrement of whether exogenous molecules could increase type IV collagen synthesis. skin thickness and an increment of skin properties. The analysis of integrins in rat skin by FACScan Normal human keratinocytes (mammary skin, 44-y-old subject) were grown in HK-SFM (Gibco) and the western blotting indicated that the Fucus extract stimulated the expression of 01β1 molecules. medium supplemented with EGF (5 ng per ml) and pituitary gland extract (50 µg per ml) for 24 Taken together, these results suggested that integrins play an important role to maintain skin h. The cells (25 01ϫ 103 per microplate well) were then grown in HK-SFM containing thickness and skin properties in in vivo and that the Fucus extract might regulate thickness and soyasapogenol A and B isolated from soybean seeds for 72 h. The amount of type IV collagen properties of skin due to an increment of integrin molecules. synthesized in the culture medium was determined by ELISA using a polyclonal (1:200) anti human type IV collagen antibody (Pasteur Intitute, Lyon, France). Proteins were measured with the bicinchoninic acid assay (Sigma). Collagen is expressed as ng per 100 µg protein. Soyasapogenol A and B (5–25 µg per ml) increased keratinocytes type IV collagen synthesis by 19%–38% (p Ͻ 0.01). The positive control was TGF-01β (ϩ50% at 10 ng per ml). A combination (1/1 wt/wt) of ecdysterone, an ecdysteroid that increases keratinocyte differentiation and desmosomes formation plus soyasapogenols maximized type IV collagen synthesis. These data show for the first time the specific stimulation of type IV collagen synthesis. A combination with ecdysterone may well be a most appropriate way of stimulating keratinocyte type IV collagen synthesis.

375 376 Connective Tissue Growth Factor Gene Expression in Tissue Sections from Eosinophilic Fasciitis Sarpogrelate Hydrochrolide Regulates Collagen Metabolism in Human Dermal Fibroblasts N. Hayashi, T. Kakinuma, K. Kikuchi, G. Grotendorst and K. Tamaki M. Ono, K. Watanabe, K. Omori and H. Ueki Department of Dermatology, University of Tokyo, Tokyo, Japan and Department of Cell Biology Department of Dermatology, Kawasaki Medical School, Kurashiki, Japan and Anatomy, University of Miami, Miami, Florida Sarpogrelate hydrochloride (SPG), a serotonin receptor antagonist, is widely used in the treatment Connective tissue growth factor (CTGF) is a cystein-rich peptide that stimulates fibroblast of vascular obstructive diseases. Recently, SPG has been applied in treatments of the ischemic proliferation and extracellular matrix synthesis in a manner similar to transforming growth factor- change and Raynaud’s phenomenon in patients with scleroderma. In this study, to understand 01β (TGF-01β). CTGF is exclusively produced by skin fibrlblasts after activation by TGF-01β. biological effects of SPG for clinical application in scleroderma, we investigated effects of SPG on A recent study revealed that TGF-01β gene expression was demonstrated in fascia with eosinophilic the collagen metabolism in cultured human fibroblasts. Normal human dermal fibroblasts were fasciitis (EF). In order to clarify CTGF involvement in fibrogenesis of eosinophilic fasciitis, We cultured with 50 µM SPG for 48 h, then the expressions of the type-1 collagen and the matrix investigated CTGF gene expression in tissue sections of eosinophilic fasciitis using none-radioactive metal loprotease-1 (MMP-1) were analyzed. The results showed that SPG upregulated MMP-1 in situ hybridization. Significant CTGF mRNA was detected in the fascia which had remarkable expression 7-fold at mRNA level, 10-fold at enzymic activity but downregulated type-1 collagen inflammatory cell infiltrations. The inflammatory cells consisted of eosinophils, lymphocytes and 7-fold at mRNA level. Similar effects were observed at 1 µM SPG for 2 wk culture which is histiocytes. Moderate message was observed in the deep dermis which had moderate inflammation. clinically administrated concentration. These effects, however, was not competitive with serotonin Cases which had no inflammation did not show any CTGF message. Cases of CTGF positive EF or SPG did not affect to the intracellular level of the inositol-1, 4, 5-triphosphate in fibroblasts. demonstrated elevation of serum aldolase which is a useful indicator of disease activity. Our finding In addition, there are no evidence showing that dermal fibroblasts express 5-HT2 (5-hydroxytrypta- suggests that CTGF gene expression is demonstrated when an active fibrotic change is maintained mine) receptor. Our study suggests that SPG may act not only for vasodilation but also against in EF, and is an available indicator of disease activity. collagen accumulation via an unique pathway in patients with scleroderma.

377 378 Mechanical Force Alters Urokinase Plasminogen Activator Levels in Human Dermal Fibroblasts Effect of Colchicine on Fibroblast Matrix Metalloproteinase I Activity E. M. McIlrath, W. Filsell, G. Jenkins and M. R. Green S. Arumugam, I. Aronson and R. Kumar Biosciences Division, Unilever Research Colworth, Bedford, UK Department of Dermatology, University of Illinois College of Medicine, Chicago, Illinois In skin, fibroblast behaviour may be modulated by mechanical force changes in the surrounding We have previously reported the use of colchicine in the treatment of lipodermatosclerosis (LDS), ECM. We have therefore examined how mechanical force alters levels of the dermal proteases which is characterized by inflammation and fibrosis in areas of venous insufficiency. LDS lesional responsible for ECM breakdown. Urokinase plasminogen activator (uPA) is a serine protease that tissue reveals inflammation, perivascular fibrin, active collagen I synthesis, increased but imbalanced catalyses the conversion of plasminogen into plasmin. Plasmin, in turn, converts a range of matrix expression of matrix metalloproteinases (MMP) MMP-1, MMP-2 and their tissue inhibitors metalloproteinases into their active forms; hence uPA may act as a control point for tissue (TIMP). Serum studies of these patients also show increased levels of plasminogen activator degradation in vivo. We have used the contracting collagen gel model to compare uPA levels in inhibitor-1 and decreased levels of tissue plasminogen activator, suggesting an alteration in finely fibroblasts subjected to high and low tensional forces. Gene expression of uPA, uPA receptor controlled process of fibrinolysis. Using enzyme zymography the effect of varying concentrations (uPAR) and PAI-1 (uPA main physiological inhibitor) was analysed in cells embedded in attached of colchicine (known to induce collegenase and inhibit collagen production) on production of (high tension) and contracting (low tension) gels by competitive PCR. Compared to the MMP-1 and MMP-2 were studied on confluent fibroblast cell cultures. Here we report that corresponding attached gels, expression of uPA and uPAR in contracting gels increased 2-fold 4 colchicine specifically increases the activity of MMP-1 but has no effect on MMP-2. The clinical h after gel detachment. However, expression of PAI-1 also increased (2.6-fold) 4 h after gel response in LDS to colchicine may in part be due to its role in the differential regulation of detachment. uPA protein was examined by fibrin overlay zymography. Levels increased in MMPs. Maintaining a delicate balance between the various MMPs may be a critical factor in contracting gels with maximal increase (1.7-fold) 1 d after gel detachment. In addition, we perserving tissue homeostasis in LDS. examined the effect of uPA inhibitors on collagen gel contraction. Contraction was inhibited by amiloride and p-aminobenzamidine, suggesting that uPA may be involved in the gel contraction process. Taken together, these data suggest that dermal fibroblasts respond to altered tensional forces in the contracting collagen gel model by changing uPA levels and that uPA plays a role in gel contraction. 586 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

379 380 Analysis of Basement Membrane Formation and Epidermal Differentiation After Grafting of Downregulation of Collagen Synthesis in Cultured Fibroblasts by Pyridoxal Benzoyl Hydrazone Cultured Epithelial Sheet onto Wound Sites of Abraded Scar H. Yang, S. Murad and S. Pinnell S. Amano, K. Kadoya, M. Tsunenaga, S. Inomata, . H. T. Nishiyama and N. K. Shiseldo* Division of Dermatology, Department of Medicine, Duke University Medical Center, Durham, Research Center, Yokohama, Japan; *Department of Plastic and Reconstructive Surgery, School North Carolina of Medicine, St. Marianna University, Kawasaki, Japan Pyridoxal benzoyl hydrazone (PBH), the active form of the fibrobsuppressive agent benzoic acid Autologous transplantation of cultured human keratinocyte sheets has been used clinically for hydrazide, inhibits the synthesis of collagen and proliferation of human skin fibroblasts in culture. improving disfigured skin surfaces, such as wide areas of burn scars, tattoos, depigmentation and To investigate the possibility that PBH might inhibit collagen synthesis at a pretranslational level, so on. However, little is known about the histology of the grafted sites. We have examined the we measured the relative amount of collagen mRNA in cells treated with PBH and two of its deposition of basement membrane components and the expression pattern of differentiation inactive analogs, pyridoxal isonicotinoyl hydrazone (PIH) and pyridoxal phenylacetyl hydrazone markers in the epidermis of biopsy specimens from the early stage until the stable stage after (PPH). Total RNA was isolated from cells treated for 72 h with 100 µM ascorbic acid and 50 µM grafting, by means of immunohistochemistry. Cultured epithelial sheets were grafted onto the PBH, PIH, or PPH, then hybridized with 32P-labeled cDNA probes specific for pro01α1(I)collagen wound sites remaining after abrasion of mature scar tissue. High grafting efficiency was obtained mRNA and 28S ribosomal RNA in a northern blot analysis. Compared to ascorbate-treated and the appearance of the burn scar was improved. Laminin 5 was stained strongly at the dermal- controls, the level of collagen mRNA was found selectively decreased 68% in cells treated with epidermal junction by 6 d after grafting. Type IV collagen was not seen until day 7, but strong PBH but not in those treated with PIH or PPH. Of the two species of pro01α1(I)collagen staining was observed at day 10 after grafting. Type VII collagen was faintly observed even 2 wk mRNA, only the 4.8 kb transcript normally present in larger amount than the 5.8 kb transcript post-grafting. After 3 wk, each component was strongly stained at the dermal–epidermal junction. was affected by PBH. The reduced level of procollagen mRNA in PBH-treated cells reflected The deposition of basement membrane components did not depend on the degree of scarring at the inhibition of collagen synthesis measured by collagenase digestion of [3H]proline-labeled graft recipient sites. Expression of the differentiation markers, filaggrin and involucrin was faintly proteins. The tight coupling of procollagen synthesis and secretion known to exist in cell culture observed up to 7 d after grafting. After 10 d, filaggrin was expressed only in the granular layer of lead us to suggest that the collagen suppressing effect of PBH may be related to its ability to the epidermis, as in normal skin. However, involucrin was observed in the whole spinous layer inhibit prolyl hydroxylation by iron chelation resulting in the synthesis of unhydroxylated and the granular layer from 10 d until several months after grafting, in contrast to the situation in procollagen, a nontriple helical and thus nonsecretable form containing the inhibitory propeptides. normal skin, where it is expressed only in the upper spinous layer and the granular layer. These results suggest that the grafting of autologous cultured epithelial sheet onto the wound site after the abrasion of scar tissue can improve the appearance of the disfigured skin surfaces, but the process of differentiation in the grafted epidermis is not exactly the same as that in normal skin.

381 382 Role of Extracellular Matrix Components in Basal and Squamous Cell Characterization of Dermal Collagen in C3H Mice at Different Stages of Hair Cycle Carcinomas K. Yamamoto and M. Yamauchi M. Lahiri and G. Stricklin Dental research center, University of North Carolina, Chapel Hill, North Carolina, and Sunstar Division of Dermatology, Vanderbilt University, Nashville, Tennessee Inc., Osaka, Japan To explore the ECM–tumor interaction in basal (BCC) and squamous (SCC) cell carcinomas, Hair follicles develop or regress in accordance with their hair cycle. Our previous studies indicated human early passage BCC (10), SCC (three) and one baso-squamous carcinoma (BSC) cell lines that the remodeling of collagen was more active at anagen stage than that at telogen (Yamamoto were established and grown on the ECM components fibronectin and laminin. The conditioned K. and Yamauchi M., J Invest Dermatol 1996). The purpose of this study was to further characterize media collected were assayed for the matrix metalloproteinases (MMP) MMP-1(ELISA), MMP- and compare dermal collagen at these two hair cycle stages. Skin samples were obtained from 11- 2 and MMP-9 (gelatinase substrate gels). Apoptotic cells (annexin Vϩ) were also determined. wk-old C3H mice at anagen (n ϭ 5), which was induced by shaving, and telogen stages (n ϭ 5). Preliminary data show substantial production (62–186 ng per ml) of MMP-1 by one of 10 BCC, The samples were pulverized and the dermal collagen was first extracted with 1 M NaCl containing three of three SCC, and one BSC plated on fibronectin. Unlike normal keratinocytes that show neutral solution and then 0.5N acetic acid. Based on the hydroxyproline content in each extract, inhibition of MMP-1 by laminin, two of 10 BCCs and two of three SCCs grown on laminin the amount of neutral salt soluble collagen in anagen skin was significantly higher than that of produced significant levels of MMP-1 (75–400 ng per ml) that were much higher (6- and 4-fold, telogen (2.7% Ϯ 0.6% in anagen and 1.3% Ϯ 0.5% in telogen, n ϭ 4). Another aliquots of respectively) than when grown on fibronectin. Three of 10 BCCs, two of three SCCs, and one pulverized samples were subjected to pepsin digestion (Ͼ95% solubilized) and type I collagen was BSC released MMP-9 in the presence of laminin as compared to five of 10 BCC, one of three fractionated by salt precipitation method. Then 01α1 and 01α2 chains of type I collagen were SCC and one BSC in the presence of fibronectin. MMP-2 production was not altered by laminin separated by SDS-PAGE, transferred onto a PVDF membrane and subjected to amino acid analyses or fibronectin. Minimal apoptosis was detected on laminin (0.22%, 0.18%, 0.08% of cells in BCC, to evaluate the hydroxylation of proline and lysine residues on the respective chains. The results SCC, and BSC, respectively) or fibronectin (2.2%, 0.29%, 0.9%, of cells in BCC, SCC, and BSC, demonstrated that the levels of lysine hydroxylation on both 01α1 and 01α2 chains of type I respectively). These findings suggest that the MMP expression pattern in BCCs and SCCs may collagen obtained from anagen skin were both significantly higher than those of telogen (5.0% vary from that of normal keratinocytes. higher in 01α1 chain and 15.6% higher in 01α2 chain, n ϭ 5). The proline hydroxylation on both chains of type I collagen was also found to be slightly higher in anagen skin than that of telogen. These results indicate that skin at anagen stage contains more newly synthesized collagen than that of telogen and that they are post-translationally distinct from those of telogen. These characteristics of collagen in anagen stage might be important to facilitate an environment around hair follicles for their migration and growth.

383 384 Effect of Nifedipine on Procollagen Type 1 C-Peptide in Normal Human Dermal Fibroblasts Cultured Skin Substitutes with Fibroblasts Express Basement Membrane Antigens and Ultrastruc- R. Yavel, E. Goyart,* T. Mammone,* Y. P. E, W. Lee and A. Shalita tures In Vitro State University of New York Health Science Center at Brooklyn and Biological Research S. Boyce,*† A. Supp* and G. Warden*† Division, *Estee Lauder Co., Melville, New York, New York *Shriners Burns Hospital and †Department of Surgery, University of Cincinnati, Cincinnati, Ohio Calcium channel blockers are a chemically heterogeneous group of compounds which recently Efficacious healing after transplantation of cultured human keratinocytes (HK) is limited by poor have been shown to modulate the extracellular matrix formed by human vascular smooth muscle attachment, and consequently poor engraftment, blistering and mechanical instability. To address cells and normal human dermal fibroblasts (NHDF). NHDF were grown to confluence in these limitations, cultured HK were inoculated on to collagen-based substrates that contained 0 microtiter wells and were treated for 60–72 h with nifedipine and verapamil at various or 5 01ϫ 105 human fibroblasts (HF)/cm2 to generate cultured skin substitutes (CSS). After 11 d concentrations to determine: (i) the effect of these calcium channel blockers on the in vitro incubation with air exposure, samples were analyzed for collagens IV and VII, and laminin 5 by production of collagen by NHDF using a simple enzyme immune assay (EIA) for Procollagen immunohistochemistry (IHC), and for basement membrane ultrastructure by transmission electron Type 1 C-Peptide (PIP), the major collagen component in the dermal extracellular matrix; (ii) microscopy (TEM). IHC showed strong linear staining for each antigen in CSS with HF, and no qualitatively whether such a change is evident at the transcription level using gene amplification organized staining of antigens without HF. Similarly, ultrastructure of basement membrane was PCR reaction. The primers used were specific for the pro01α 1 chain of type 1 procolllagen observed in CSS with HF only. Histiotypic ultrastructures included lamina densa, anchoring fibrils, cDNA.We found a statistically significant increase in the production of PIP for NHDF treated and hemidesmosomes. These structures correspond, respectively, to collagens IV and VII, and with nifedipine at all concentrations tested (1 01ϫ 10–6–1 01ϫ 10–9 M) when compared to PIP laminin 5. In addition, the epithelial organization of cultured keratinocytes in the presence of production for untreated cells (p Ͻ 0.05). Moreover, it appeared to have a dose-dependent effect fibroblasts resembled closely the anatomy of epidermis. Without fibroblasts, keratinocytes were on PIP production; there was a 9.95% and a 34.26% increase in production for nifedipine at a poorly organized. These results suggest that keratinocytes recognize, organize and synthesize final concentration of 1 01ϫ 10–9 and101ϫ10–6 M, respectively. PCR results indicate that the anatomic structures as a function of the presence of fibroblasts in skin substitutes. Although the upregulation in collagen production may occur at the transcription level. Our study indicates that mechanisms for these responses by keratinocytes are not fully understood, these results demonstrate calcium channel blockers are capable of upregulating collagen production in NHDF and that that greater anatomic fidelity of cultured skin substitutes to native human skin can be obtained nifedipine in particular appears to upregulated the pro01α 1 chain at the transcription level. by combination of fibroblasts with keratinocytes. These results are consistent with the clinical However, these results are not sufficient to determine the overall effects of nifedipine and verapamil observation that CSS with keratinocytes and fibroblasts rapidly reform complete basement on extracelluar matrix (ECM) remodeling, since ECM remodeling also reflects the activities of membrane, and regenerate stable epidermis that does not blister after grafting. matrix metalloproteinases and tissue inhibitor of metalloproteinases (TIMP) as well as the influence of a variety of cytokines and growth factors. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 587

385 386 Endoglin, a Transforming Growth Factor P Receptor III, is Upregulated in Scleroderma Platelet-Derived Growth Factor (PDGF) Induces Matrix-Bound Molecules that Stimulate Migra- Dermal Fibroblasts tion of Keratinocytes in Skin Equivalents M. Connolly, K. Venstrom, D. Finlay, X. Dou, J. Jackman and P. Findell J. Teumer, J. Dahlquist, D. Whitston, G. Downing and L. Xu Department of Dermatology, UCSF, San Francisco, California; and Roche Bioscience, Palo Organogenesis Inc., Canton, Massachusetts Alto, California PDGF is known to stimulate cell proliferation, growth factor release, and extracellular matrix Scleroderma fibroblasts have been shown to have an aberrant phenotype characterized by the deposition. Keratinocytes or fibroblasts that overproduced PDGF-AA were used to construct skin overproduction of a variety of extraceflular matrix molecules. A role for TGF-01β in mediating equivalents (SEs). Overexpression of PDGF stimulated fibroblast proliferation in skin equivalents. the upregulation has been proposed. Upregulation of the TGF-01β receptors I and II has been In SE with PDGF-fibroblasts there was an increase in fibroblast number throughout the matrix. previously reported in scleroderma fibroblasts, however, no information about TGF-01β RIIIs, In PDGF-keratinocyte SEs, fibroblast number was increased only in the matrix covered by endoglin or beta-glycan, has been reported. We analyzed eight scleroderma fibroblast cell lines epidermis and not in peripheral matrix not covered by epidermis, suggesting that PDGF from and four normal controls by an oligonucleotide microarray GeneChip analysis. To validate the keratinocytes did not diffuse through the matrix. method, we compared TIMP-3 production and found it elevated among scleroderma patients Using a serum-free culture system, the epidermis migrated over the entire surface of the matrix compared to controls, as previously reported. Transcripts for TGF-01β3 and TGF-01β receptors containing PDGF-fibroblasts. In contrast, the epidermis in control as well as PDGF-keratinocyte I and II were present, but no significant differences between normal and scleroderma fibroblasts SEs did not spread beyond the region upon which it was applied. Thus, PDGF stimulated were identified. No transcripts for beta-glycan, one of the TGF-PRIIIs, were detected in any epidermal migration but only on matrix exposed to excess PDGF. This stimulation was not from sample. In contrast, 318 fibroblast cell lines had increased endoglin transcripts compared to no the direct action of PDGF on keratinocytes since keratinocytes did not express PDGF01α receptors detectable levels of transcripts in the four controls. Endoglin is a 180-kDa homodimeric protein but rather must have been from some indirect activity of PDGF. Conditioned medium from that binds TGF-01β1 and TGF-01β3. We confirmed our GeneChip findings by western blot PDGF-fibroblast cultures or SEs could not induce the epidermal migration in uninfected SEs or analysis and immunofluorescent tissue staining. Eighty per cent of our scleroderma fibroblasts had in keratinocyte migration assays. By semiquantitative RT-PCR, no increase was observed in α β elevated levels of endoglin compared to 20% of normal controls at the protein level. Increased mRNA levels of TGF01 , TGF01 1–3, aFGF, bFGF, or KGF. Thus, these growth factors were tissue staining of blood vessels in involved scleroderma skin was also seen. The increased expression not responsible for inducing the epidermal migration. Collectively, the observations suggested that of endoglin correlated with clinical fibrosis. This is the first report of a TGF-01β RIII being epidermal migration was induced by a matrix-bound factor, such as an extracellular matrix increased in scleroderma and gives indirect support for the role of TGF-01β in mediating this molecule. The results demonstrate the ability of matrix to influence keratinocyte behavior aberrant tissue phenotype. independent of the action of soluble growth factors.

387 388 A New Model for Studing Re-Epithelization of Partial Thickness Wounds in Hypoxic Skin Effect of Anagen Induction on the Re-Epithelization of Partial Thickness Wounds T. P. Sullivan, S. C. Davis, E. I. Sanders, A. L. Cazzangia, W. H. Eaglstein, G. W. Elgart and P. E. I. Sanders, G. W. Elgart, T. P. Sullivan, S. C. Davis, A. L. Cazzaniga, R. Paus, W. H. Eaglstein M. Mertz and P. M. Mertz Department of Dermatology and Cutaneous Surgery, University of Miami School of Medicine, Department of Dermatology and Cutaneous Surgery, The University of Miami School of Medicine, Miami, Florida Miami, Florida Oxygen is necessary for several critical events in wound healing, e.g. hydroxylation of lysine to Partial thickness wounds involve the epidermis and a portion of the dermis. These wounds heal proline and the subsequent release of collagen from fibroblasts, epithelialization, angiogenesis, and from the wound margins and from the appendages, which include the hair follicles. Hair follicles the ability of leukocytes to kill bacteria. We have developed a model for hypoxic wound healing undergo cycling, from a proliferative stage (anagen) to an involutional stage (catagen) to a resting in pigs, an animal whose skin and healing closely resembles human. Eight flaps were outlined on stage (telogen). We hypothesize that iatrogenic synchronized induction of the anagen phase the dorsal flank of a pig. Using an electrokeratome, partial thickness wounds (10 01ϫ 701ϫ0.3 in skin prior to wounding may effectively enhance the rate of re-epithelization of partial mm) were created on the distal, medial and proximal portions of all eight outlined flaps. Only thickness wounds. four of the eight marked flaps were actually incised. The nonincised flaps served as our nonischemic Hair follicles on one side of the dorsal skin of a pig were induced into anagen using creme wax controls. The flaps were 6 cm in width by 9 cm in length. The incised flaps were created by depilation. The contralateral side was not waxed and served as the control. Using an electrokeratome, incising along both sides and along the bottom (distal) edge leaving the top edge intact. The flap partial thickness wounds (10 01ϫ 701ϫ0.3 mm) were created on both sides of the animal. was then cut away from the underlying tissue. Once this was completed the flap was returned to Excisional biopsies were taken of the wounds on days 3, 5, and 7 postwounding to assess the its bed and stapled in place. On days 3, 5, 7, and 10 post-wounding, pO2 measurements were extent of re-epithelization. made using a transcutaneous, noninvasive oxygen monitor. In addition, excisional biopsies of the Blinded histological assessments were made by measuring the percentage of re-epithelization wounds were taken at these time points. Measurements of pO2 taken from the surgical flaps observed in depilated skin versus control skin. Biopsies were evaluated by day postwounding (i.e., showed marked hypoxemia when compared to the control flaps. The control flaps demonstrated days 3, 5, and 7 postwounding) and the mean percentage of re-epithelization was calculated. a consistent pO2 of 40–45 on days 0–10. The distal segments of the surgical flaps had an average Results are as follows: day 3, 60% Ϯ 9% (depilated) vs 48% Ϯ 12% (control); day 5, 98% Ϯ 3% pO2 of 14, the medial segment a pO2 of 26, and the proximal segment a pO2 of 38. Furthermore, (depilated) vs 89 Ϯ 1% (control). Results from day 5 were statistically significant with a p ϭ delayed re-epithelialization was observed in the ischemic flaps compared to controls as shown by 0.016. Complete re-epithelization of both the depilated and control skin (i.e., 100%) was seen by mean percentage re-epithelialization. Results are as follows: day 3, 10% (ischemic) vs 21% (control); day 7. We will further evaluate these findings with the use our NaBr Assay which allows evaluation day 5, 21% (ischemic) vs 21% (control); day 7, 64% (ischemic) vs 93% (control). Complete re- of the entire wound surface for complete versus incomplete re-epithelization. epithelialization was seen in both groups by day 10. We plan to further refine our model for use in the evaluation of healing in hypoxic wound environments.

389 390 Deletions of the PTCH Gene in Sebaceous Nevi Detection of Loss of Heterozygosity on Chromosome 9q22.3 in Microdissected Sporadic H. Xin, D. Matt, G. Burg and R. Bo¨ni Trichoepithelioma Department of Dermatology, University Hospital, Zu¨rich, Switzerland D. Matt, H. Xin, A. O. Vortmeyer, Z. Zhuang, G. Burg and R. Bo¨ni Sebaceous nevi are congenital malformations of the skin and may develop into basal cell carcinoma. Department of Dermatology, University Hospital, Zu¨rich, Switzerland To date, the molecular basis for its carcinogenic potential remains unknown. In sporadic BCC Trichoepithelioma (TE) occurs either in multiple lesions or as a solitary lesion. Multiple TE is frequent allelic deletions at the BCC susceptibility gene, human homolog of Drosophila patched transmittet as an autosomal dominant trait and the gene has been mapped to chromosome 9p21. (PTCH) on chromosome 9q22.3 have been demonstrated. Solitary TE occurs more commonly than multiple TE and is not inherited. Histologically, TE The objective of this study was to test whether allelic deletion of the PTCH gene could be contain horn cysts, abortive hair papillae as well as areas with the appearance of basal cell carcinoma detected in sebaceous nevi. (BCC). In sporadic BCC frequent deletions at the BCC susceptibility gene, human homolog of Twenty-one paraffin-embedded sebaceous nevi were investigated in this study. Basaloid cells in Drosophila patched on chromosome 9q22.3 have been demonstrated. conjunction with mature sebaceous glands as well as epidermal layer apart from sebaceous nevi The objective of this study was to test whether loss of heterozygosity (LOH) on either 9p21 (the were microdissected and subjected to single step DNA extraction. Analysis was perfomed with gene for multiple familial TE) or on chromosome 9q22.3 (BCC susceptibility gene) could be the polymorphic markers D9S15, D9S252, D9S287 and D9S303. Of the 20 informative sebaceous detected in archival sporadic TE. nevi, eight (40%) exhibited loss of heterozygosity at least at one locus. We found one case showing We studied 29 randomly selected sporadic TE by microdissection and polymerase chain reaction loss of heterozygosity at all four loci, two cases with loss at three loci and two cases with loss at using paraffin-embedded, formalin-fixed material on glass slides. Analysis was performed with the two loci. polymorphic markers IFNA and D9S171 (9p21) as well as D9S15, D9S303, D9S287 and Here, we provide the first evidence of the involvement of the tumor suppressor gene PTCH in D9S252 (9q22.3). sebaceous nevi. Our study supports on a molecular basis the clinical observation, that a subset of LOH at 9q22.3 was identified in 12 of 29 cases (41%) with at least one marker, while LOH was sebaceous nevi may develop into basal cell carcinoma. retained at 9p21. The results show that BCC LOH can be frequently identified in paraffin embedded sporadic TE after routine processing and indicate a common gatekeeper mechanism for TE and BCC. 588 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

391 392 CD44 Variant Isoform CD44v10 Expression of Human Melanoma Cell Lines is Upregulated by Cloning and Sequencing of a cDNA Encoding a Serine Protease Homologous to Human Hyaluronate and Related to Migration Complement C1r Precursor from an Allografted Mice Skin and Its Expression in E. coli. C. Yoshinari, S. Horiuchi, H. Byers and T. Akasaka T. Kim,* S. Byun, J. Hyun and Z. Ryoo Departments of Dermatology and Biochemistry, Iwate Medical University, School of Medicine, Department of Dermatology, College of Medicine and Research Institute of Animal Science, Morioka, Iwate, Japan and Department of Dermatology, Boston University School of Medicine, Catholic Research Institute of Medical Science, the Catholic University of Korea, Seocho-gu, Boston, Massachusetts Seoul, Korea CD44, a multifunctional adhesion receptor involved in cell–cell and cell–matrix interactions, plays Many genes are involved in the rejection of a skin allograft. Expressions of these genes, which an important role in local progression and metastasis of malignant tumors. We investigated CD44 have not been thoroughly explored, are controlled by interactions of the host immune system variant isoform mRNA in six human melanoma cell lines and determined cell migration on and the grafted skin. In order to identify differentilly expressed genes in an allografted mice skin, hyaluronic acid (HA) coated substrates. Hematopoietic form (CD44H) mRNA expression increased we used DDRT-PCR analysis. Eighteen candidate clones were selected by DDRT-PCR anaysis in all melanoma cell lines after plating on HA, whereas the CD44 variant exon 10 (CD44v10) and one of these clones has a high homology with human complement C1r. To obtain full-lengh mRNA increased in only three of the cell lines. Cell migration rates increased on substrates coated cDNAs, a mouse skin cDNA library was screened by this clone probe. From DNA sequence with HA in the three CD44v10 positive cell lines, however, the three CD44v10 negative cell analysis, the screened cDNA insert which contains 5’noncoding, 2121 nucleotides coding for a lines showed no modification in migration rates. Immunofluorescent labeling of CD44v10 revealed polypeptide precusor of 707 amino acids, and 3’noncoding region was shown to span 2416 plaques localized to the periphery of cells. Cell lines with increased CD44v10 plaques exhibited nucleotides of mRNA, not containing a polyA tail. The coding region of the full-lengh cDNA significantly higher mean migration rates on HA. These results indicate that CD44v10 expression was cloned in E. coli expression vector and expressed a protein of about 85 kDa similar to that of functionally relates to melanoma cell migration and suggests CD44v10 and HA interaction plays human. The complement system is composed of highly sophisticated chain reactions which enable a role in variable tissue invasion and aggressiveness of different melanoma clones. a host to discriminate between self and nonself. Recognition of nonself organisms occurs via the classical pathway by interactions between C1 and various antibodies. The result obtained in this work suggests that the clone encoding a serine protease homlogogous to human complement C1r precursor is involved in the complement reaction of the rejection of the skin allograft.

393 394 Differentiation-Related Gene Expression in Human Cultured Keratinocytes during Autocrine The Mitochondrial Electron Transport Chain Serves a Signaling Role in Ceramide-Induced Growth Conditions. Upregulation of Involucrin Expression by Retinoic Acid Keratinocyte (KC) Intercellular Adhesion Molecule-1 (ICAM-1) Expression Y. Poumay,* P. Smits,† F. Herphelin* and M. Pittelkow‡ I. Felsner, S. Grether-Beck, M. Berneburg, H. Schmitt and J. Krutmann *Department Histology-Embryology, University of Namur, Namur, Belgium; †Laboratory of Clinical and Experimental Photodermatology, Department of Dermatology, University of Du¨ssel- Molecular Biotechnology, UIA, Wilrijk, Belgium; and ‡Department of Dermatology, Mayo Clinic, dorf, Germany Rochester, Minnesota The mitochondrial (mt) electron transport chain is part of the signal transduction pathway that With the development of keratinocyte cell culture, the control of epidermal differentiation has controls gene expression in human cells. Accordingly, depletion of the mt electron transport was been progressively defined at the cellular and molecular levels. Under autocrine growth (low shown to inhibit tumor necrosis factor 01α (TNF01α)-induced gene expression in human calcium medium concentration and absence of serum and growth factors), keratinocytes induce fibrosarcoma cells. The underlying mechanism is not known, but recent studies indicate that expression of suprabasal keratins 1 (K1) and 10 (K10) at culture confluence. We have investigated ceramides, which serve a signaling role in TNF01α-induced gene expression, can directly affect mRNA expression of epidermal genes in identical culture conditions at high cell density mitochondria and by doing so induce the generation of reactive oxygen species. Ceramide (postconfluence), or after treatment with sodium butyrate (NaBu), phorbol ester (TPA) or retinoic signaling is also critically involved in ultraviolet A radiation (UVAR)-induced expression of acid (RA). Postconfluent keratinocytes downregulate K1 and K10 mRNA expression but ICAM-1 in human KC. In the present study we have therefore asked whether ceramide-induced upregulate involucrin when compared with early confluent cells. At subconfluence, treatment for ICAM-1 expression in human KC was mediated through the mt electron transport chain. Normal 24 or 96 h with 1 mM NaBu inhibits keratinocyte growth but only slightly upregulates involucrin human KC as well as KB cells were stimulated with C6-ceramide and subsequently assessed for expression. At confluence, NaBu treatment decreases K1 and K10 mRNA levels without affecting ICAM-1 mRNA expression by semiquantitative, differential RT-PCR. Stimulation of KC with involucrin expression. Treatment at subconfluence with 1–10 ng per ml TPA inhibits cell growth, C6-ceramide induced ICAM-1 mRNA expression in a dose-and time-dependent manner. upregulates involucrin and transglutaminase but does not induce K1 or K10 expression. Together, Prevention of the electron flow between complex II of the mt respiratory chain and ubiquinone our results confirm that K1 and K10 expression is only initiated by cell confluence/stratification, through addition of nontoxic concentrations of the specific inhibitor thenoyltrifluoroacetone but repressed during late stages of differentiation. Treatment of confluent keratinocytes with 10– (TTFA) completely abrogated C6-ceramide-induced KC ICAM-1 expression. The role of 8–10–5 M RA inhibits K1 and K10 expression similar to other reported culture conditions, but mitochondria in ceramide-induced KC ICAM-1 expression was further analyzed by using Rho- interestingly, we show for the first time that RA upregulates involucrin during autocrine growth O cells which are deficient in oxidative phosphorylation. In Rho-O cells, strong ICAM-1 conditions. These findings parallel in vivo effects of RA which modulate epidermal proliferation, expression was induced by rh interferon-01γ, whereas in the same experiment, C6-ceramide differentiation and expression of selected gene products. stimulation did not increase ICAM-1 expression above background levels. Taken together these observations provide strong evidence for a signaling role of the mt electron transport chain in ceramide-, and possibly UVAR-induced KC gene expression.

395 396 TNF01α and IL-101α Induce Suprabasal Expression of Keratin K6 in Normal Human Skin Multiple Drug Resisance -Related Messenger RNA Expression in Malignant Melanoma Before M. Komine, N. Hattori, K. Tamaki, M. Blumenberg and I. M. Freedberg and After Chemotherapy Department of Dermatology, The University of Tokyo, Tokyo, Japan; and NYU Medical Center, N. Ichitashi and Y. KitaJima New York, New York Department of Dermatology, Gifu University of School of Medicine, Gifu, Japan Among the inflammatory keratins, K6 is one of the most extensively expressed keratin proteins. Malignant melanoma is notoriously resistant to chemotherapeutic agents, but the exact mechanisms We have identified the regulatory element of TNF01α and IL-101α which consists of C/EBP involved in this drug resistance are unknown. One recently defined major mechanism involves binding sites in the promoter of keratin K6 gene, which needs NFkB and C/EBPb for the the overexpression of multiple drug resistance related protein (MRP). In order to determine the induction by TNF01α. Its regulation at the protein level was not fully investigated because of the relationships between this MRP and clinical chemoresistance of malignant melanoma, we examined strong keratin K6 expression of cultured keratinocytes even in the unstimulated state. This study alteration of mRNA levels by RTPCR analysis of RNA from formalin-fixed paraffin-embedded is to demonstrate the induction of keratin K6 by TNF01α and IL-101α at the protein level and sections. Dewaxed and Hematoxyli ne-stained sections (10 micrometer) were microdissected to to elucidate its distribution in vivo. We utilized normal human skin samples incubated with or separate mass of tumor cells from normal tissues. RNA was extracted from the sectiones using without cytokines, and performed immunohistochemical staining with anti-K6, as well as C/ proteinase K and STAT 60. Total RNA (0.5 microgram) was subjected to RT-PCR using two EBPb, and NFkB antibodies. TNF01α caused suprabasal induction of keratin K6 and induced oligonucleotide primers sets specific for human MRP and one primer set specific for betaactin suprabasal nuclear translocation of NFkB, but there was no change in the expression of C/EBPb. used for a housekeeping gene as a contorol. Primers used for amplification of the MRP gene Keratin K6 expression in the lesional psoriatic skin, squamous cell carcinoma and adnexal apparatus were 5’ACG GTC GGG GAG ATT GTC AAC 3Ј and 5’GCC CAG ATT CAG CCA CAG is well correlated with the overexpression of C/EBPb, but the perilesional expression of keratin GAG Y, yielding an expected amplificate of 135 bp; and those for amplification of the beta-actin K6 is focal in spite of the diffuse overexpression of C/EBPb. This is the first report of TNF01α gene were 5Ј ACC CAC ACT GTG CCC ATC TAC 3Ј and 5Ј CAT CTC CTG CTC GAA and IL-101α inducing keratin K6 at the protein level, which strongly supports our previous data GTC CAG Y, yielding an expected aniplificate of 204 bp. These primers were newly designed of transcriptional regulation of keratin K6. in this study. We compared ratios of MRP to betaactin in six recurrent cases of melanoma before and after chemotherapy. These six cases of melanoma were found to express MRP to a certain extent even before chemotherapy. Five of these six recurrent melanoma after chemotherapy (dacarbazine, nimustine, vincrisrinc, and interferon beta) showed a rise of MRP mRNA. These results indicate that a significant level of MRP is intrinsically present in malignant melanoma before exposure to drugs and it increases after chemothcrapies, leading to a suggestion that MRP play a role in increasling chemoresistanec in malignant melanoma. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 589

397 398 Relationship Between p53 Status and Amount of hsp72 Induction in Human Cancer Cells Expression of Antimicrobial Peptides in Keratinocytes: Beta Defensin 2, a Molecular Marker for A. Tsubakimoto,* K. Ohnishi,† A. Takahashi,† M. Hatoko,* T. Muramatsu,* T. Shirai* and the Inflamed State of Human Skin T. Ohnishi† L. Poquet, P. Ancian, M. Rivier, M. Cavey, A. Clucas and J. Voegel Departments of *Dermatology and †Biology, Nara Medical University, Kashihara City, Nara, Japan GALDERMA R&D, Sophia Antipolis, France We have previously reported that heat sensitivity of cancer cells depends on p53 status using Keratinocytes produce antimicrobial peptides which defend human skin against bacterial and human glioblastoma cells and mouse fibroblast cells transfected with different type p53 gene. In fungal infection. We studied the expression levels of beta defensin 1, beta defensin 2 (hBD2) and this study, we further investigated the contribution of p53 status to heat-induced accumulation of cathelicidin related antimicrobial peptide by semiquantitative RT-PCR in cultured human hsp72 in human glioblastoma (A-172), human melanoma (HMV1) and human squamous cell keratinocytes (NHKs). hBD2 basal expression was not modulated after treatment with RAR, carcinoma of tongue (SAS) having wild-type p53. We transfected mutant p53 (mp53) to those RXR, PPAR, VitD3 and GR receptor ligands. hBD2 mRNA levels in NHKs were strongly cell lines and the dominant negative nature of mp53 gene was used for the analysis of the p53 induced by P. aeruginosa, phorbol ester and lipopolysaccharide. To confirm in vivo the induction function. Western blot analysis showed that p53 protein was accumulated after heating much of hBD2 observed under inflammatory conditions in vitro, hBD2 mRNA levels were studied in more in mp53-transfected cells than in nontransfected cells. These results suggested that p53 may psoriatic skin. hBD2 mRNA levels were induced 7-fold in involved compared to noninvolved regulate heat sensitivity or heat tolerance through the regulation of hsp72 induction. skin samples. In skin biopsies of psoriasis patients treated with the glucocorticoid Betamethasone, a correlation between a decrease of hBD2 mRNA levels and an improvement of the clinical score could be observed. In summary, hBD2 expression is strongly induced in keratinocytes under inflammatory conditions. It may therefore be used to follow the course of inflammatory skin diseases and to screen for novel anti-inflammatory agents.

399 400 Use of High Density cDNA Arrays to Monitor Differential Gene Expression in Reconstructed Epi- Mapping of the Psoriasis Vulgads-Susceptible Gene dermis M. Ezuka, A. Ozawa, J. Sugai, Y. Sasao, K. Iwashita, Y. Kawakubo, M. Ohkido and A. Oka,* P. Ancian, M. Lenoir, V. Quekenborn and S. Michel G. Tamiya,* T. Shfina,* M. Tomizawa,* K. Iwata,* H. Inoko,* M. Ohta,† Y. Katsuyama† GALDERMA R&D, Sophia-Antipolis, France Department of Dermatology, Tokai University School of Medicine; *Department of Molecular Various methods have been developed in order to detect and quantify differential gene expression, Life Science, Tokai University of School of Medicine; †Department of Medicine, Shinsyu including differential display, SAGE display and high density cDNA arrays (HDA). In this study, University, Japan we set up a HDA method to determine the effect of 1, 25-dihydroxy vitamin D3 on the gene We have determined the genomic sequence of the 2.0 Mb entire HLA class I region from the expression profile of reconstructed epidermis treated by gamma interferon (IFN01γ). Commercially MICB to HLA-F genes. This region includes susceptible loci for various diseases such as psoriasis available membranes (Atlas Cancer, Clontech) containing 588 different cDNAs were used. Around vulgaris. In order to identify causative gene for psoriasis vulgaris, association analysis by microsatellite 50% of the spotted cDNAs were detected using, as a template, 5 µg of total RNA prepared from marker was carried out based on the obtained sequence data in 76 Japanese patients with psoriasis reconstructed epidermis. The observed mRNA expression profile was highly reproducible and a vulgaris and 132 healthy controls. The results suggests that the pathogenic gene for psoriasis 2-fold variation in mRNA level was considered to be significant. IFN01γ treatment of reconstructed vulgaris is narrowed down within 110 kb surrounding the OTF3, SCI and S genes. Among 12 epidermis induced modification of the expression of close to 10% of the genes studied, including markers, five alleles displayed statistical significance. Especially, there were two alleles remarkably specific IFN01γ induced genes, and IL-17. In addition, the expression levels of HLA-DR01α, associated with patient. ICAM-1 and VCAM-1 was determined by semiquantitative RT-PCR analysis. Our results show that 1, 25-dihydroxy vitamin D3 inhibits the expression of genes associated with inflammation which were induced by IFN01γ in reconstructed epidermis.

401 402 Mutation of Cysteine Residues 369, 552, and 687 in Lysyl Hydroxylase 1 Eliminates Enzyme Tissue Specificity of a New Splice Form of the Human Lysyl Hydroxylase 2 Gene Activity L. C. Walker and H. N. Yeowell M. A. Overstreet, L. C. Walker, S. Murad and H. N. Yeowell Division of Dermatology, Department of Medicine, Duke University Medical Center, Durham, Duke University Medical Center, Durham, North Carolina North Carolina We have established the contribution of each of the 10 cysteine residues in lysyl hydroxylase 1 Lysyl hydroxylase (LH) is an essential enzyme in collagen biosynthesis that hydroxylates lysine (LH1) to the activity of LH1, an important enzyme that hydroxylates lysine residues required in residues required in the crosslinking that gives collagen its structural integrity. This is the first the crosslinking that gives collagen its tensile strength. Initially we identified a 5 amino acid (aa) report of alternative RNA splicing in a gene for LH in a normal population. This splicing event, deletion (DLCRQ) between residues 367–371 in two patients with Ehlers–Danlos syndrome type which we have observed in the LH2 gene, appears to be tissue specific. The recently reported VI (EDS VI) who are compound heterozygous for the mutations in the LH gene responsible for sequence of the LH2 isoform from a human kidney cDNA library was predicted to encode a 737 their LH deficiency. We hypothesized that the loss of the central cysteine residue (C369) was amino acid (aa) protein. In the present study, we have isolated a cDNA for LH2 from human responsible for their dimished enzyme activity. This was confirmed by the synthesis of mutant skin fibroblasts that codes for a protein of 758aa, of which 21aa are encoded by a new exon. This forms of LH1 in which C369 and the nine other cysteines in the enzyme were individually 63 bp exon, designated exon 13A, is located between exons 13 and 14 of the originally described mutated to serine by site-directed mutagenesis of a normal pAcGP67/LH1cDNA construct LH2 gene. PCR amplification of cDNAs between exons 13 and 14 gave two distinct LH2 mRNA followed by expression in an Sf9 insect cell/baculovirus system. For each construct, SDS/PAGE populations. A 209-bp transcript was expressed in mRNAs isolated from all tissues examined and and western analysis showed that a mutant enzyme of the correct size (85 kDa) was secreted in was the only transcript expressed in skin, lung, aorta and dura, whereas in mRNAs from spleen, this system. As observed in the EDS VI patients, the loss of C369 eliminated the activity of the cartilage, liver, kidney, frontal lobe and placenta, an additional shorter 146 bp transcript was expressed enzyme and, moreover, the mutation of residues C552 and C687 also completely amplified. Direct sequencing showed that these two mRNAs resulted from the alternative splicing inactivated the enzyme. The C375S mutation produced a significant, but not complete, decrease of exon 13A. The transcript containing exon 13A is expressed as the major LH2 form in all tissues in activity, whereas mutations of C267, C270, C566 and C680 had only a minor effect. In except kidney or spleen. Analysis of genomic DNA from skin, placenta and spleen showed that contrast, the C204S and C484S constructs had normal levels of activity. Although with the both transcripts were generated from the same LH2 gene. In a developmental study only single exception of C204, each of the cysteines is conserved between the three human isoforms of this transcripts which included exon 13A were amplified from normal fetal skin at different stages of enzyme (LH1, LH2 and LH3), as well as chick and rat LH1, the relative contribution of each gestation, suggesting that although exon 13A is variably expressed in different tissues, this alternative cysteine to LH activity may be structurally determined by, for example, disulfide bond formation splicing event is not developmentally regulated. in a particular location of the enzyme. 590 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

403 404 Requirement of an AP-1 Site in the Calcium Response Region of the Involucrin Promoter Identification of ‘‘Fingerprint Genes’’ of Dendritic Cell Maturation Using GeneArray Technology D. C. Ng, S. Shafaee, D. Lee and D. D. Bikle M. Mohamadzadeh, F. Hui, P. Bergstresser and A. Takashima Endocrine Unit, Veterans Affairs Medical Center, University of California, San Francisco, California Department of Dermatology, UT South-western Medical Center, Dallas, Texas Involucrin is a major protein of the cornified envelope of keratinocytes that provides much of Maturation of dendritic cells (DC) represents a critical transition from the cells specialized for the structural integrity of the skin. Elevated extracellular calcium induces the gene expression of antigen uptake and processing into cells that deliver T cell-stimulatory signals. A number of this differentiation marker in cultured human keratinocytes. A 3.7-kb fragment of this gene features that accompany DC maturation have been induced experimentally by different stimuli, contains the necessary elements to drive a luciferase reporter in a calcium-dependent manner. The including bacterial lipopolysaccharide (LPS). We have reported previously that the murine DC purpose of this study is to characterized this gene for the presence of calcium-dependent elements. lines, XS52 and XS106, both respond to LPS treatment by elevating surface expression of CD86 We have sequenced the upstream region of the involucrin promoter and localized a calcium and MHC class II molecules and by secreting pro-inflammatory cytokines. As an initial step in response element that contains an AP-1 site (TGAGTCA). Mutation of this site abolished the untangling the complex molecular events in DC maturation, we sought to identify the profiles of promoter activation by calcium. Compared to cells grown in 0.03 mM calcium, the binding genes that are either induced or down-regulated during LPS-triggered maturation of XS52 and activity of factors within nuclear extracts from keratinocytes for this AP-1 site was enhanced XS106 DC lines. For this aim, we employed the Gene Discovery Array (GDA) membrane, on three-fold in cells grown in 1.2 mM calcium. Immuno-electrophoretic mobility shift (supershift) which 18,650 different genes are printed. Poly Aϩ RNAs were isolated from XS cells before and assays identified JunD, c-Fos, FosB, Fra1, Fra2 as factors that bind to the AP-1 element. Western after a 16-h incubation with 100 µg per ml LPS and then reverse transcribed to prepare cDNA analysis of the proteins in the nuclear extracts showed that the levels of these factors increased probes. GDA membranes were hybridized with the 33P-labeled probes and then analyzed by the with calcium treatment. Calcium and phorbol-myristate acetate (PMA) enhanced the involucrin phospho-imager. Digitized data were then processed using a special program, Array Vision, to promoter synergistically in a protein kinase C (PKC)-dependent manner. In addition to the more compare the expression levels of individual genes among the four groups; i.e. LPS-stimulated distal AP-1 element, both the calcium and the PMA actions of the keratinocytes require the XS52, untreated XS52, LPS-stimulated XS106, and untreated XS106. Expression levels remained proximal region of the involucrin promoter, containing a proximal AP-1 site, for optimal activity. relatively unchanged (Ͻ2-fold difference) by LPS in Ͼ90% of the analyzed genes, indicating that Therefore, the AP-1 factors in keratinocytes are essential for the activation of the involucrin hybridization conditions were comparable between LPS-treated and nontreated groups. We promoter by calcium. identified 42 and 76 genes that were markedly (Ͼ5-fold) upregulated by LPS in the XS52 and XS106 lines, respectively. Of these genes, 10 genes were, in fact, upregulated Ͼ5-fold in both DC lines; they included the calmodulin, procollagen 01α2 (I) chain precursor, and nuclear ribonuclear protein L genes. We also identified 32 and 286 genes that were markedly (Ͼ5-fold) downregulated by LPS in the XS52 and XS106 lines, respectively. Of these genes, four genes were downregulated Ͼ5-fold in both DC lines; they included the phosphoenolpyruvate carboxykinase and S-II transcription factor genes. Based on these observations, we propose that DC maturation is characterized by induced or abrogated expression of selected groups of genes. Identification of these ‘‘fingerprint genes’’ now provides a unique opportunity to study DC maturation at the molecular level.

405 406 Tissue-Specific Expression of KET and its Splicing Variant CUSP, Members of the P53 Family Moderation of Phenotypic Severity in Dystrophic and Junctional Forms of Epidermolysis Bullosa T. Egbert, A. Marchbank, R. Dellavalle, J. DeGregori, L-J. Su, P. Walsh and L. Lee Through Alternative Splicing of Exons Containing Nonsense or Frameshift Mutations University of Colorado School of Medicine, Denver Health, and VA, Denver Colorado J. McGrath, G. Ashton, J. Mellerio, J. Salas-Alanis,* O. Swensson,† E. Christophers,† J. McMillan Several groups, including ours, have recently identified a third gene closely related to the p53 and R. Eady tumor suppressor and its newly discovered homologue p73. This gene, mapped to chromosome Department of Cell and Molecular Pathology, St John’s Institute of Dermatology (GKT), St 3q, was originally named KET, but has also been identified as CUSP, p40, p51, p63 and p73 L. Thomas’ Hospital, London, UK; *Ciencias Biologicas, Departamento de Biologia, Universidad KET has multiple splice variants that can theoretically create up to 18 different transcripts. CUSP Autonoma de Nuevo Leon, Monterrey, Mexico; †Department of Dermatology, Christian Albrechts is the major KET transcript of keratinocytes. University, Kiel, Germany Studies of the tissue distribution of KET have conflicting results. To define KET expression, Nonsense mutations on both alleles of the genes encoding type VII collagen (COL7A1) or laminin multiple tissue northern blots were probed using a consensus sequence that hybridizes to all known 5 (LAMA3, LAMB3, or LAMC2) usually result in severe forms of recessive dystrophic or KET transcripts and also a sequence specific for the CUSP N-terminus. Indirect IF was performed junctional epidermolysis bullosa (RDEB, JEB), respectively. In this study we assessed two unrelated on rat organs using antibodies to CUSP. A comparison of our results (columns 1 & 2, CUSP & families whose mutations in genomic DNA predicted severe RDEB or JEB phenotypes but in KET) to those of others is shown below. Our studies examine both protein and mRNA expression whom the manifestations were milder than anticipated. The RDEB patients had a homozygous for CUSP and mRNA for KET, while the other studies are almost entirely restricted to frameshift mutation in exon 19 of COL7A1 (2470insG). Clinically, there was generalized blistering mRNA expression. but only mild scarring. Skin biopsy revealed positive type VII collagen immunoreactivity and recognizable anchoring fibrils. The JEB patients were compound heterozygotes for frameshift/ nonsense mutations in exons 3 and 17 of LAMB3 (29insC/Q834X). These patients did not have CUSP KET rat KET p51 p40 p73 L p63 the lethal form of JEB but, as adults, displayed the milder generalized atrophic benign EB variant. There was undectectable laminin 5 staining at the dermal-epidermal junction using an antibody skin ϩϩ ϩND ND ND ϩ to the 01β3 chain, but faintly positive 01α3 and 01γ2 chain labeling, and there was variable thymus ϩϩ ϩϩ– ϩϩ hypoplasia of hemidesmosomes. To explain the milder RDEB and JEB phenotypes in these skeletal muscle – ϩ – ϩ ND ϩ ND families, RT-PCR, using RNA extracted from frozen skin, was able to provide evidence for some heart – weakly ϩ – ϩ ND – ϩ rescue of mutant mRNA transcripts with restoration of the reading frame. In the RDEB patients, lung – weakly ϩ – ϩ ND – ND transcripts containing in-frame skipping of exon 19 of COL7A1, or exons 19 and 20, or a 474- kidney * weakly ϩ ––ϩ–ϩ bp deletion in the cDNA were detected. In the JEB patients, transcripts with an in-frame deletion testis –– ––– ϩϩ of exon 17 of LAMB3 were identified. The truncated proteins encoded by these transcripts lack certain domains involved in cell-matrix attachment but may still provide some adhesion. Elucidation of mutations in DEB and JEB has proved useful in establishing prognosis but this study shows the *CUSP expression in kidney was restricted to capsular epithelium. ND ϭ not done limitations of relying solely on mutation analysis of genomic DNA and emphasizes the importance In conclusion, the CUSP splicing variant has very restricted tissue expression. Detection of KET of immunohistochemistry, electron microscopy and mRNA assessment as parallel investigations. but not CUSP mRNA in skeletal muscle, heart, and lung indicates either that KET variants are expressed in disparate locations, or there are as yet unidentified additional p53-like genes that hybridize to the KET probe. These studies support the hypothesis that the p53-like proteins are expressed in a distribution distinct from that of the ubiquitously expressed p53 and have functions that are tissue-specific.

407 408 A Novel Point Mutation Affecting the Function of the C1-Inhibitor in a Patient with Type H Papular Atrichia Associated with Retardation of Bone Age is Caused by a Novel Homozygous Hereditary Angioneurotic Edema Missense Mutation in the Human Hairless Gene Y. Miyashita, M. Ono, M. Ono and H. Ueki R. Kruse, S. Cichon, M. Anker, G. Weinlich, M. Schmuth, P. Fritsch, P. Propping and M. No¨then Department of Dermatology, Kawasaki Medical School, Kurashiki, JapanC1 inhibitor (C1-INH) Institute of Human Genetics, University of Bonn, Germany; Department of Dermatology, plays important roles in the classical pathway of the complement system and the regulation of University of Innsbruck, Austria vascular permeability through the kinin-forming system. Hereditary angioneurotic edema (HANE), Papular atrichia is an autosomal recessive disorder defined by universal congenital alopecia and which inherited as an autosomal dominant trait, is due to deficiency of the C1-INH. The disease disseminated papular lesions. As shown recently, papular atrichia is allelic to isolated universal is characterized by reccurent episodes of subcutaneous and submucosal edema. congenital alopecia, both caused by a mutation in the human hairless (HR) gene. Here we report We studied the molecular genetic basis of C1-INH deficiency in a patient with type II HANE on the molecular genetic study in a South Tirol family affected with papular atrichia and retardation by nucleotide sequencing. A single base change (G01→A) at nucleotide 16830 in the eighth exon of bone age. Typing with microsatellite markers supported linkage to the HR locus at chromosome was presented in the C1-INH gene. This mutation converted the codon for Valine 458 to 8p21–22. This prompted us to sequence all coding exons and all exon-intron boundaries of the Methionine. In order to understand whether this point mutation caused dysfunction of the C1- HR gene in one affected individual. We identified three homozygous nucleotide changes leading INH, we synthesized mutant C1-INH based on the point mutation using an in vitro transcription/ to amino acid substitutions at different sites. The three homozygous changes were present in all translation system. Mutant C1-INH was then reacted with the C1 esterase and its inhibition other affected family members available. In order to identify the one disease-causing nucleotide activity was analyzed by a fluorogenic substrate for the C1 esterase. In result, inhibition activity change we determined the population frequencies of each variant by genotyping 606 unrelated of mutant C1-INH against the C1 esterase was 85.2% reduced as compared with normal C1- control individuals. We identified two of the three nucleotide changes (Leu526Pro at nt. 1577 in INH, indicating that this point mutation affects the function of the C1-INH and may become exon 5; Thr1022Ala at nt. 3064 in exon 15) to be frequent HR polymorphisms and an AO˜ G the causative agent for type flHANE. missense mutation at nt. 2909 in exon 14 leading to an 01Asn01→Lys substitution (codon 970) as the disease-causing mutation responsible for this papular atrichia phenotype. This molecular finding supports the idea that the HR gene is not only involved in hair development but also in the process of ossification. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 591

409 410 Enhanced Expression of Biologically Active Vascular Endothelial Growth Factor in Genetically Genetic Association Between the MHC S Gene and Susceptibility to Psoriasis Modified Cultured Skin Substitutes D. M. Supp, A. P. Supp, S. Bell and S. T. Boyce R. T. Ahnini, M. Cork, N. Camp,* J. Mee,† S. Keohane, G. Duff and F. di Giovine Shriners Burns Hospital and Children’s Hospital Research Foundation, Cincinnati, OhioCultured Division of Molecular and Genetic Medicine, University of Sheffield, UK; *Genetic Epidemiology, skin substitutes (CSS) have been used as adjunctive therapies in the treatment of burns and chronic LDS Hospital, Salf Lake City; †Centre for Dermatology, UCL wounds, but they are limited by lack of a vascular plexus. This deficiency leads to greater time Psoriasis is a common inflammatory skin disease which has been shown to be associated with the for vascularization compared to native skin autografts and contributes to graft failure. Genetic MHC region. The strongest HLA association is found between familial early onset psoriasis and modification of CSS to enhance vascularization could hypothetically lead to improved wound HLA-Cw6. However, the involvement of the HLA-C gene in the disease pathogenesis is not healing. To address this hypothesis, human keratinocytes were genetically modified by transduction clear, and it cannot be excluded that other genes in the HLA region are functionally important. with a replication incompetent retrovirus to overexpress the gene encoding vascular endothelial The MHC S gene, located 160 kb telomeric of HLA-C is expressed specifically in keratinocytes growth factor (VEGF), a specific and potent mitogen for endothelial cells. CSS consisting of in the terminal phases of differentiation. Two single-nucleotide polymorphisms in the gene were collagen-glycosaminoglycan substrates inoculated with human fibroblasts and either VEGF- analysed, at positions ϩ619 and ϩ1243. DNA was collected from 235 psoriatic patients and 374 modified or control keratinocytes were prepared, and were cultured in vitro for a period of 3 wk. ethnically matched controls, and HLA-Cw6, S(ϩ619) and (ϩ1243) allelic distributions studied by Northern blot analysis demonstrated enhanced expression of VEGF in genetically modified PCR-RFLP genotyping based on previously published methods (Steinle et al, 1992; Ishihara et al, keratinocytes and in CSS prepared with modified cells. Furthermore, the VEGF-modified CSS 1996). As expected, a significant association was found between psoriasis and HLA-Cw6 [OR ϭ secreted greatly elevated levels of VEGF throughout the entire culture period. The bioactivity of 7.75 (95% c.i. ϭ 5.68, 10.56)]. No association was found between disease (or any subtypes by VEGF secreted by the genetically modified CSS was demonstrated using a microvascular endothelial age at onset) and the S gene polymorphism at position ϩ619 despite its close proximity to HLA- cell growth assay. VEGF-modified and control CSS were grafted to full-thickness wounds on the C and the strong linkage disequilibrium between the loci. A significant association considered as flanks of athymic mice, and enhanced VEGF expression could be detected in the modified grafts trend (P ϭ 201ϫ10–9) was detected between the rarer allele at MHC S (ϩ1243) and psoriasis for at least 1 wk after grafting. These results indicate that genetic modification of keratinocytes in in the overall dataset [OR ϭ 2.66 (1.86, 3.82)]. This effect was most pronounced in early onset CSS can lead to increased VEGF expression which could prospectively improve vascularization psoriatic [OR ϭ 3.43 (2.21, 5.32)]. Also, homozygosity for the associated allele at MHC S of CSS for wound healing applications. (ϩ1243) increased the risk of disease over that for carriage of HLA-Cw6 alone [OR ϭ 9.38 (5.99, 14.70)]. Analysis of data suggested that the association of (ϩ1243) allele 2 (rarest allele) with psoriasis was secondary to that of HLA-Cw6. Allele 2 of (ϩ 1243) thus provides an additional risk in psoriasis susceptibility. The strong association found here, coupled with the biological involvement of the MHC S gene product (corneodesmosin) in skin physiology, may be related to the impaired desquamation characteristics of psoriatic lesions.

411 412 Engineering a New Autocrine Loop by Genetic Modification of Human Melanocytes: Hepatocyte Fibroblast Growth Factor Receptor 3 Mutations Associated with Acanthosis Nigricans Result in Growth Factor Expression Stimulates Proliferation Abnormal Bcl-2 Expression in Keratinocytes J. Morgan and K. Hamoen G. A. Bellus, J. Reichel, E. Whalen and D. A. Norris Center for Engineering in Medicine and Surgical Services, Massachusetts General Hospital and Department of Dermatology, University of Colorado, Denver, Colorado Harvard Medical School, Shriners Burns Hospital, Boston, Massachusetts Missense mutations in the fibroblast growth factor receptor 3 (FGFR3) gene are known to cause several human skeletal dysplasias (e.g. achondroplasia) as well as at least two craniosynostosis Hepatocyte growth factor (HGF) is a fibroblast-derived protein that affects growth, motility and syndromes. Early childhood onset and persistence of acanthosis nigricans (AN) is a prominent differentiation of epithelial cells and has also been shown to be a mitogen for human melanocytes. feature of some of these disorders but is not related to insulin resistance. Two FGFR3 HGF has recently been implicated as an important factor for the growth and dissemination of mutations have a strong correlation with AN [i.e. FGFR3 K650M: SADDAN syndrome (severe melanomas. To investigate the potential role of HGF in the progression from melanocyte to achondroplasia with developmental delay & acanthosis nigricans) and FGFR3 A391E: Crouzon melanoma cell, we have used retroviral-mediated gene transfer to introduce the gene encoding syndrome with acanthosis nigricans (CAN)] but do not share other phenotypic features. In order human HGF into normal human epidermal melanocytes, thus causing these cells to produce a to determine how these two mutations affect keratinocyte gene expression we have performed an growth factor they normally do not express and creating a new autocrine loop as sometimes immunohistochemical analysis of skin samples from two patients who are heterozygous for either occurs in melanoma cells. Modified cells synthesized and secreted significant levels of HGF in vitro. the K650M or A391E mutation. Keratins K5, K10, FGFR3 and Bcl-x were expressed normally Cells overexpressing HGF had a higher rate of proliferation and formed large, dense, melanin in SADDAN and CAN as well as in obesity related AN (ORAN) which is presumably due to positive colonies on a plastic surface when compared to unmodified cells. Immunohistochemical hyperinsulinemia. Expression of EGFR, FGFR1, FGFR2 and FGFR4 were significantly upregul- staining showed HGF positive melanocytes with varying levels of expression and HGF protein ated in SADDAN but not in CAN or ORAN. In addition, abnormal expression of K18, K19 was detected throughout the whole cell. These results suggest that overexpression of HGF may and involucrin were observed in the basal layer of SADDAN skin. Expression of Bcl-2 was play a role in the multistep transformation of a melanocyte into a melanoma cell. Using this gene localized to the basal layer in normal skin and ORAN but was found throughout all the epidermal transfer approach as well as the transplantation of modified melanocytes as part of a skin equivalent layers in SADDAN and CAN. These results indicate that certain FGFR3 mutations may specifically to athymic mice may help us to gain a better understanding of the role of HGF in the pathogenesis interfere with keratinocyte apoptosis and suggest a mechanism for generating the abnormal of melanoma. epidermal findings in FGFR3 related AN.

413 414 Introduction of Integrin Binding Motifs into Recombinant Feline Parvovirus for Re-targeting to Gene Delivery to HaCaT Cells and Primary Keratinocytes with a Parvovirus LuIII Gene Vector Human Melanoma Cells J. Corsini, F. Maxwell, G. Bellus and I. H. Maxwell I. H. Maxwell, J. Chapman, F. Maxwell and J. Corsini Department of Dermatology, University of Colorado Health Sciences Center, Denver, Colorado Department of Dermatology, University of Colorado Health Sciences Center, Denver, Colorado LuIII rodent parvovirus is a small, nonenveloped, single-stranded DNA virus that we are developing Parvoviruses are small, single-stranded DNA viruses that possess antitumor activities. One member into a vehicle for delivery of therapeutic genes into human cells, including keratinocytes. We of this family, the feline panleukopenia virus (FPV), does not normally infect human cells, so is a have observed efficient transduction of the HaCaT (human keratinocyte) cell line with recombinant good candidate for modifications designed to re-target recombinant FPV bearing therapeutic genes virus expressing the luciferase gene from either the cytomegalovirus immediate early promoter or into human tumor cells. We have previously shown that the capsids of recombinant FPV can from the LuIII P4 early promoter. The transduced HaCaT cells continued to express luciferase at support small peptide insertions without disrupting the virus. As an approach to targeting a low level for at least 10 passages (1/30 dilutions), suggesting that the recombinant genome is melanomas, many of which aberrantly express 01αV01β3 and 01αV01β5 integrins, we have able to persist in these cells. Inefficient transduction of primary keratinocytes by two rodent introduced integrin (01αV01β3 and 01αV01β5) binding motifs into the coat of recombinant parvoviruses closely related to LuIII has been reported, so it was of interest to assess the ability of luciferase-FPV. One such RGD virus specifically and efficiently transduced the luciferase gene our LuIII vectors to transduce primary keratinocytes. To this end, four isolates of human foreskin into a human rhabdomyosarcoma line that expresses 01αV01β3 and 01αV01β5 integrins, but was keratinocytes were transduced with a P4 luciferase virus and were observed to efficiently express unable to transduce human melanoma lines expressing these integrins. While the ability to re- luciferase for at least 3 d. Furthermore, in several experiments, infection by wild type LuIII lysed target to a human tumor line provides a ‘‘proof-of-principle’’, the inability to re-target human primary keratinocytes, again in contrast with published studies of a rodent parvovirus closely melanomas expressing the appropriate integrins indicates that integrin presence is necessary but related to LuIII. These data provide a basis for further investigation of the potential use of rodent not sufficient for the re-targeting process. Identification of required cofactors present in the parvoviruses in gene therapies that target keratinocytes. rhabdomyosarcoma line, but presumably absent or inactive in the melanoma lines, is the goal of future work. 592 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

415 416 Novel PTEN Mutations in Three Families with Bannayan–Zonana Syndrome Implications of the Position of Glycine Substitution Mutations in COL7A1 in Determining J. Tok Celebi, H. Tsou, F. Chen, H. Zhang, X. Ping and M. Peacocke Dystrophic Epidermolysis Bullosa Phenotype Department of Dermatology, Columbia University, New York, New York J. Mellerio, G. Ashton, R. Mohammedi, N. Whittock, M. Dunnill, R. Eady and J. McGrath Germline mutations in PTEN, a putative tumor suppressor gene, has been identified in two Department of Cell and Molecular Pathology, St John’s Institute of Dermatology (GKT), St autosomal dominant inherited hamartoma syndromes, Cowden syndrome (CS) and Bannayan– Thomas’ Hospital, London, UK Zonana syndrome (BZS). BZS is characterized by macrocephaly, central nervous system abnormalit- Almost all cases of autosomal dominantly inherited dystrophic epidermolysis bullosa (DEB) arise ies, lentigines of the penis, and hamartomatous growths including lipomas, intestinal polyposis, from glycine substitution mutations within the triple helical domain of the type VII collagen gene, and vascular malformations, whereas CS is identified by skin findings and hamartomas in a variety COL7A1. These mutations cause perturbations in type VII collagen synthesis and/or secretion, of tissues, with an increased risk of breast and thyroid cancers. While both diseases exhibit distinct resulting in morphological and functional abnormalities of anchoring fibrils. However, a number phenotypic features, the presence of macrocephaly, lipomas, and intestinal polyposis in both of glycine substitutions have also been described which are silent in the heterozygous state but conditions suggests a partial clinical overlap. Moreover, the coexistence of features common to result in recessive DEB when inherited in trans with a pathogenetic mutation on the second CS and BZS within the same family has also been observed. To date, nine families with BZS COL7A1 allele. To investigate this disparity further, we determined COL7A1 gene mutations in have been screened for PTEN mutations, of which five were found to exhibit mutations in this 95 patients with different forms of DEB using PCR of genomic DNA, heteroduplex analysis and gene. We identified three novel germline mutations in the PTEN coding sequence from three direct nucleotide sequencing, and identified 14 different glycine substitution mutations. Ten of unrelated families with the BZS phenotype. We report a de novo 4 bp deletion at nucleotide 983 these resulted in dominant disease (G1522E, G1776R, G1791E, G2006A, G2040D, G2043R, in exon 8, 983delAAAT, which results in a premature stop at codon 342. The second mutation G2046 V, G2242R, G2369S and G2713R). Of note, the mutation G2043R was identified in is a splice site mutation in intron 3209 ϩ G01→A, identified in the proband and the affected four unrelated dominant DEB patients, in two of whom it had arisen as a de novo event. In mother. Finally, the third mutation we report is a de novo C01→T transition at nucleotide 1003 addition, four silent glycine substitution mutations were identified in patients with recessive DEB in exon 8, which results in arginine being replaced by a stop codon, R335X. While all three (G1703E, G1782R, G2375S, and G2674D) who were compound heterozygotes for a nonsense mutations are novel in BZS, the nonsense mutation, R335X, has been identified in three unrelated or splice site mutation on the second COL7A1 allele. Of the dominant mutations, four lie within families with CS. Of interest, we found R335X mutation in a single kindred with members exon 73 of COL7A1, whereas the silent glycine substitutions are within distant collagenous exhibiting both CS and BZS phenotype. The notion that CS and BZS may be within the segments of the triple helix. The nature of the amino acid substitution does not appear to have spectrum of the same primary disorder awaits further studies. an effect on the pathogenicity of the mutation. Rather, the postition of mutations within the triple helix as a whole or within individual collagenous segments appears to be more significant in determining the phenotypic sequelae. The clinicopathological consequences of the glycine substitution mutations identified in this study increase our understanding of the relative functional importance of different regions of the type VII collagen triple helix.

417 418 Phenotypic Findings of Cowden Syndrome and Bannayan–Zonana Syndrome in a Family Touching the Untouchable: The Use of Antisense Technology in Skin Associated with a Single Germline Mutation in PTEN I. Freedberg, M. Blumenberg and M. Tomic-Canic P. Lee, J. Tok Celebi, H. Tsou, F. Chen, H. Zhang, X. Ping, M. Lebwohl, J. Kezis and M. Peacocke The Ronald O. Perelman Department of Dermatology Department of Dermatology, Columbia University, New York, New York NYU Medical School, New York, New York Cowden syndrome (CS) and Bannayan–Zonana syndrome (BZS) are two hamartoma syndromes Oligonucleotides and their analogs are powerful tools with multiple uses, ranging from basic with distinct phenotypic features. Although partial clinical overlap exists between CS and BZS, research to therapeutic intervention. Arguably, skin is the most appropriate target tissue for such they are considered separate entities. CS is characterized by facial papules, oral papillomatoses, and tools. The modern antisense technology can be used to target the molecular basis of disease as hamartomas in the breast, thyroid, endometrium, and gastrointestinal tract. In addition to benign well as to answer directly basic scientific questions. Therefore, we have developed a set of novel tumors, individuals with CS are at increased risk of developing breast and thyroid cancers. approaches to apply the antisense technology to skin research. Using antisense oligonucleotides Characteristic features of BZS include macrocephaly, CNS abnormalities, lentigines of the penis, we have mapped the TNF01α signal transduction cascade as well as the glucocorticoid and and hamartomatous growths such as lipomas and intestinal polyposis; however, there is no known retinoic acid receptor regulatory pathways in epithelial cells. We used antisense oligonucleotides, associated risk of malignancy. Linkage studies have identified PTEN as the susceptibility gene for synthesized as phosphorotioates, to target specific mRNAs and eliminate the corresponding both disorders, suggesting allelism. We present a germline nonsense mutation, R335X, in PTEN, proteins from the regulatory pathway. Using such approach we have found that proteins TRAF2, in a family consisting of two female members with the phenotypic findings of CS and two male IKK, C/EBP01β, NF01κB and I01κB are essential for the induction of K6 keratin expression by members with the phenotypic findings of BZS. Direct sequencing analysis of the PCR product TNF01α. Furthermore, histone acetyl tranferase and the nuclear receptor coactivators play a role flanking exon 8 revealed a transition of C-to -T at nucleotide 1003, resulting in a change from in suppression of epidermal keratin gene expression. Conversely, we have found that histone arginine to a stop codon in all four affected individuals in this family. The mutation was confirmed deacetylase and the nuclear receptor corepressors play a role in activation of epidermal keratin by allele-specific oligonucleotide analysis. To our knowledge, this is the first report that demonstrates gene expression. Taken together, these results break the current dogma of the role of nuclear the presence of distinct individuals with CS and with BZS in a single family associated with a receptor coregulators in transcriptional control by nuclear receptors. Knowledge gained using single germline PTEN mutation. Our data demonstrates that a single mutation in PTEN can antisense inhibition as a precise tool for functional genetic analysis will enable us to develop novel, confer susceptibility to two distinct phenotypes and suggest that breast and thyroid cancer highly specific therapeutic agents and find biochemical targets for drug development in skin. susceptibility may be found in individuals in BZS families associated with PTEN mutations.

419 420 Transduction of Human Keratinocytes with a Lentiviral Vector Splice Mutations in Xeroderma Pigmentosum Group C DNA: Intron Retention, Exon Skipping U. Ku¨hn and J. C. Vogel and Internal Neoplasms Dermatology Branch, NCI, National Institutes of Health S. G. Khan, T. D. Schneider and K. H. Kraemer Persistent gene expression has frequently been a problem in gene therapy due in part to inadequate National Cancer Institute, Bethesda, Maryland introduction of genes into slow dividing stem cells. Unlike traditional retroviral vectors that target We studied the XPC gene in five xeroderma pigmentosum (XP) group C (XPC) patients with dividing cells, the new lentiviral vectors are able to deliver genes into nondividing cells. While multiple skin cancers and defective DNA repair. Amplification of the 3.5 kb XPC cDNA by RT- retroviruses require mitosis for dissolution of the nuclear membrane to achieve genomic integration, PCR showed abnormal bands with all five cell lines. In three patients there was an insertion of lentiviral vectors have at least two accessory genes, MA and vpr, that enable genomic integration 83 nucleotides within exon 5. Sequencing of the genomic DNA revealed a 1.5-kb intron (intron by interacting with the nuclear import machinery and mediating the active transport of the viral 5a) within exon 5 thereby splitting this region into exons 5a and 5b. The 83 bp insertion was preintegration complex through the nucleopores. The goal of this study is to determine if lentiviral found to be the 3Ј end of intron 5a and the splice acceptor sequence ended with ‘‘cag’’ in DNA vectors can transduce keratinocytes (KC) and achieve sustained gene expression. In order to from normal cells. We found single nucleotide changes: ‘‘Gag’’ in XP3BE, ‘‘cCg’’ in XP23BE, produce replication-defective lentiviral vectors, we transiently cotransfected three separate plasmids and ‘‘cGg’’ in XP24BE. These changes reduced the information content (Human Mutation 12:153– into 293T human kidney cells: a packaging plasmid (pCMV8.2) containing all structural and trans- 171, 1998) of the intron 5a splice acceptor from 17.4 bits in the normal cells, to 11.5 bits, 10 bits activating viral factors (except envelope); an envelope plasmid providing either an amphotropic and 9.3 bits, respectively, and were associated with use of a cryptic alternative splice site 83 bases (from murine leukemia virus) or the vesicular stomatitis virus G-glycoprotein (VSV-G) envelope; upstream in intron 5a. XP3BE and XP24 BE also had a mRNA species deleting exon 5b. XP14BE and a transfer vector (pHR’GFP) containing a green fluorescent protein indicator gene, which is cDNA had a 90-bp (in frame) deletion of exon 7. Surprisingly, XP14BE genomic DNA contained packaged into the lentiviral vector, and ultimately integrates into the target cell genome. At a homozygous 8 bp deletion in intron 6 splice acceptor spanning positions –12 to –19. Although different time points following plasmid transfection, viral titer in the supernatant was analyzed by the mutation was not at the extreme 3Ј end of intron 6 nevertheless it reduced the information ELISA assays of p24 Gag antigen (50–100 ng per ml) and by transduction efficiency of 293T cells. content from 11.9 to 6.2 bits. XP25BE had 3 XPC cDNA species caused by a complex Viral supernatant was taken at 48–60 h after plasmid transfection and used to transduce KC. Initial homozygous mutation in the intron 11 splice acceptor: deletion of AG at –1, –2 and insertion of studies demonstrate that primary human KC can be transduced by lentiviral vectors, pseudotyped CC between –6 and –7. This mutation reduced the information content from 5.1 to 0.3 bits. with VSV-G envelope, with a transduction efficiency of 25% by flow cytometry analysis. After Three of four XPC patients homozygous for these splice mutations have developed internal optimization of KC transduction, lentiviral vectors will be compared to traditional retroviral neoplasms [lung cancer (XP3BE), spinal cord astrocytoma (XP23BE) and malignant vectors to assess which are best at achieving long-term expression. schwannoma (XP14BE)]. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 593

421 422 Different Hair-Specific Keratin (hHb6) Mutations in Two Families with Monilethrix Genotypic and Phenotypic Mosaicism of KRT1 and KRT10 Mutations in Patients with Linear E. G. Pearce, S. K. Smith and P. E. Bowden Epidermal Naevi Department of Dermatology, University of Wales College of Medicine, Cardiff, UK P. E. Bowden, D. O. Jones and R. Marks Monilethrix is a variably expressed autosomal dominant hair disorder. Patients have hair of a Department of Dermatology, University of Wales College Medicine, Cardiff, UK beaded appearance due to periodical thinning of the hair shaft. Fragility of this unique hair causes Linear epidermal naevi (LEN) are a group of mosaic conditions characterised by eruptions that patchy dystrophic alopecia due to shaft breakage, which results in a characteristic short stubble. follow Blaschko’s lines. Some patients have warty lesions identical to epidermolytic hyperkeratosis This condition can be caused by mutations in the helix encoding regions of two hair-specific (EH) and can have children with generalised EH. Both conditions are due to keratin gene (KRT) keratins, hHb1 and hHb6. We have investigated two patients from unrelated families and found defects but to date mosaicism has only been described in KRT10. We now report a mosaic KRT1 two different point mutations in the 2B helix of hHb6 that have not been previously reported. mutation in an LEN patient. Hair samples were obtained from both patients for scanning electron microscopy and genomic This male patient had severe scaling over 80% of the body including palmar-plantar surfaces. DNA was extracted from blood samples. The helix encoding regions (1A and 2B) of hHb1 and Some regions resembled generalised EH while others had warty lesions that followed Blaschko’s hHb6 were amplified from gDNA by PCR and sequenced directly. lines. Histopathology showed suprabasal reticular degeneration and extensive hyperkeratosis with Both hair samples showed the characteristic beaded appearance. Sequencing revealed two islands of normal epidermis. The reverse was found in normal skin where regions of epidermolytic heterozygous point mutations in exon 7 of hHb6, which encodes the 2B helix. Both mutations hyperkeratosis were observed. Involved keratinocytes showed cytoplasmic clumping and perinuclear affected codon 402 (glutamic acid) which occupies position 106 in the 2B helix but the precise shelling of the keratin filament network. Genomic DNA was isolated from a 4-mm skin biopsy, alteration differed. In Patient A, the codon was changed to glutamine (GAG to CAG: E402Q) the helical encoding regions of KRT1 and KRT10 were analysed by PCR and direct sequencing. while in patient B, the change was to lysine (GAG to AAG: E402K). Unaffected relatives did not The exon 1 sequence of KRT1 was characteristic of a deletion mutation so PCR fragments of have the mutation. The 1A helix of hHb1 and hHb6 as well as the 2B helix of hHb1 were each allele were cloned into pGEM-T and sequenced independently. Codon 194 (TTC) was normal. Both mutations disrupted a Taq I restriction site and RFLP analysis produced a diagnostic absent in the mutant allele of KRT1 removing a phenylalanine residue from the 1A helix (F14D). 361 bp fragment. This increases the number of different mutations in hair-specific keratins to six The mutation was not detected in clinically normal skin and other regions of KRT1 and KRT10 and firmly establishes keratin mutations as the cause of monilethrix. were normal. This is the first KRT1 mutation to be described in LEN and the first time a keratin deletion mutation has occurred as a mosaic.

423 424 Use of a New Mutation Detection Technique to Identify Genetic Mutations in a Lamellar Fibroblast Growth Factor Receptors as Candidates for Mutation in Linear Epidermal Nevi Ichthyosis Family C. S. Munro, C. Moss and O. M. Wilkie N. V. C. Chia,* R. P. Nair* and J. T. Elder*† Southern General Hospital, Glasgow; The Childrens’ Hospital, Birmingham; and Institute of *Department of Dermatology, †Radiation Oncology (Cancer Biology), Univ of Michigan, Ann Molecular Medicine, Oxford, UK Arbor, Michigan Germline mutations in fibroblast growth factor receptors (FGFRs) 1, 2, and 3 give rise to a range Lamellar ichthyosis (LI) is an autosomal recessive skin disease characterized by dry, plate-like scales of dysmorphic syndromes including craniosynostoses and short-limbed bone dysplasia. Several all over the body and a collodion membrane at birth. The genetic defect in many cases of LI FGFR syndromes have cutaneous components, such as severe acne in Apert syndrome (FGFR2), resides in the transglutaminase 1 (TGM1) gene. The present study employed a novel and rapid cutis gyrata in Beare–Stevenson syndrome (FGFR2), and acanthosis nigrans in some types of technique to investigate possible TGM1 mutation(s). We analyzed a nonconsanguineous LI family Crouzon syndrome, thanatophoric dysplasia, and skin-skeleton–brain syndrome (FGFR3). The with one affected and two unaffected children. Using one 32P-labeled and one unlabeled primer, variety of FGFR syndromes is due to the specific effects of the causative mutations. For example, we PCR amplified the 15 exons of TGM1 gene using a deoxynucleotide mixture containing almost all cases of Apert syndrome are due to mutation in one of two adjacent codons of FGFR2, uracil and thymidine in a standardized proportion (BESS-T scan system, Epicentre Technologies). whereas mutations elsewhere in this gene give rise to Crouzon or other syndromes. If these The amplified products were digested with exonuclease 4, which cuts at the randomly incorporated mutations were to arise in embryonic life and affect only some cells of the keratinocyte lineage, uracil bases yielding two nested sets of DNA fragments equivalent to the T and A lanes of DNA they could be predicted to produce the cutaneous phenotype in a limited distribution, and to be sequencing. The remaining nucleotides were examined using the BESS-G tracker method (which manifest as epidermal nevi following the lines of Blaschko. We have recently confirmed this uses chemical cleavage to generate fragments equivalent to the G and C lanes of DNA sequencing). prediction in a patient with an acneiform linear epidermal nevus, in whom we were able to The digested products were separated on a polyacrylamide gel and the bands correlated to the demonstrate that a mutation specific to Apert syndrome (S252W in FGFR2) was present, but position of thymidine bases in the TGM1 gene. The system was validated using previously only in affected epidermis (Lancet 1998; 352:704–5). To extend this finding we have analysed a detected LI mutations (J Invest Dermatol 110:179, 1998). Both mutations from that study were series of epidermal nevi for mutations characteristic of FGFR syndromes with epidermal verified, and no false positives were found in a total of ~5000 nt scanned. In the current involvement. The lesions initially studied were verrucous epidermal nevus (two patients), nevus family, we found an Arg143Cys mutation (CGCϾTGC) in exon 3 and a Gly218Ser mutation comedonicus (two), and nevus sebaceous (one). Lesional DNA was selectively sampled by (GGCϾAGC) in exon 4 in the proband. Analysis of the parents revealed that the Arg143Cys extraction from skin scrapings taken from the nevi, and control DNA obtained from blood or mutation was maternal and the Gly218Ser mutation was paternal. The unaffected siblings did not scrapings of normal epidermis. Using specific PCR primers and restriction enzymes to amplify carry either mutation. While this work was in progress, both of these mutations were described and detect polymorphisms in regions of interest, we looked for the characteristic mutations of in the Finnish population [Am J Hum Genet 61:529, 1997]. This work demonstrates that unknown Apert syndrome (S252W and P253R in FGFR2), Crouzon syndrome with acanthosis nigricans mutations can be efficiently and reliably detected in genomic DNA without the use of (A391E in FGFR3), and (in four nevi) thanatophoric dysplasia and skin-skeleton–brain syndrome specialized equipment. (K650E and K650M in FGFR3). None of these signature mutations were detected in the nevi studied. Our negative findings do not exclude the possibility that other epidermal nevi are due to these mutations. However, the range of mutations possible in epidermal nevi may be greater than those which give rise to germline syndromes, since otherwise lethal mutations may survive by mosaicism. Hence, further analysis of growth factor receptor genes in such epidermal nevi may yet identify causative mutations.

425 426 Role of the Extracellular Signal Regulated Kinase in the Mechano-Induction of Vascular Regulation of Vascular Endothelial Growth Factor Expression in Scleroderma Fibroblasts Endothelial Growth Factor (VEGF/VPF) in Human Keratinocytes K. Kikuchi, T. Kadono, M. Kubo, H. Ihn and K. Tamaki A. Bernd, S. Kippenberger, S. Loitsch, J. Gille, M. Guschel, J. Mu¨ller and R. Kaufmann Department of Dermatology, Faculty of Medicine, University of Tokyo, Tokyo, Japan Department of Dermatology and Department of Internal Medicine, Division of Pneumology, We reported that serum levels of vascular endothelial growth factor (VEGF) in patients with University Hospital, J.W. Goethe University, Frankfurt/Main, Germany diffuse cutaneous systemic sclerosis (dSSc) were elevated compared with those of normal controls. The vascular endothelial growth factor (VEGF/VPF) is overexpressed in the epidermis of healing Cutaneous telangiectasia are often observed in affected skin of patients with dSSc. We investigated wounds. During wound formation and wound contraction the mechanical properties of the skin the regulation of VEGF expression in cultured dSSc fibroblasts (n ϭ 7) by ELISA, immunoblotting become altered. In the present study we evaluated in an in vitro attempt the relevance of mechanical and reverse transcription polymerase chain reaction. We also performed immunohistochemical stretch to induce VEGF/VPF. Human HaCaT keratinocytes were seeded on flexible silicon studies of sclerodermatous skin sections with VEGF and regulatory cytokines specific antibodies. supports and stretched longitudinally of about 10% of the initial length. After 1 h permanent Fibroblast strains derived form patients with dSSc secreted similar levels of VEGF compared with stretch the VEGF/VPF mRNA amount was increased of about 2.5-fold as detected by RT- normal fibroblasts. However, VEGF secretion was significantly more inducible by IL-1 and TGF- competitive multiplex PCR. Already after 2 h of permanent stretch the basal level of VEGF/VPF in dSSc fibroblasts. There was no significant difference of the effects of PDGF in both cell strains. became restored, underscoring the transient nature of the stimulus. On the protein level a time- Basic FGF and EGF showed no inducible effects on both strains. Expression of IL-1RI in SSc dependent mechano-induction of VEGF/VPF expression was also observed by western blot. In fibroblasts was significantly elevated. IL-1 and TGF-2 were strongly positive in inflammatory cells order to elucidate the underlying signal transduction we found a fast and transient activation of in early dSSc tissues, while only scattered cells were positive in normal controls or late fibrotic mitogen activated protein kinase (MAPK) ERK1/2, peaking already 5 min after the stretching. A tissues. VEGF and IL-1RI were positive in fibroblasts surrounding inflammatory cells in dSSc blocking of this signalling pathway by addition of the MEK1 inhibitor PD98059 abolished the tissues. These cytokine network might be important in fibrotic and angiogenic process in SSc. mechano-mediated expression of VEGF which therefore gives evidence for an ERK1/2 dependent mechano-signalling. The mechano-sensitive induction of VEGF/VPF may offers future perspectives for a clinical application in cutaneous wound closure. 594 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

427 428 Multiple Growth Factors are Involved in Growth Regulation of Dermatofibrosarcoma Protuberans Regulation of Vascular Endothelial Growth Factor Expression in Human Keratinocytes by Retinoids T. Kadono, K. Kikuchi, T. Hoashi, K. Ohara and K. Tamaki B. V. Diaz, M. Lenoir, A. Ladoux,* C. Frelin,* M. De´marchez and S. Michel Department of Dermatology, University of Tokyo, Tokyo, Japan GALDERMA R&D, Sophia-Antipolis; and *IPMC, Sophia-Antipolis, France Dermatofibrosarcoma protuberans (DFSP) is a fibrous tumor with unknown origin and its abnormal Vascular endothelial growth factor (VEGF) is highly expressed in hyperproliferative diseases such growth behavior tempts us to assume the involvement of some growth factors. Previously we as psoriasis and cancers, which are characterized by increased angiogenesis. Using normal human reported that DFSP cells overexpressed PDGF 01β receptors. In this study, we examined the role keratinocytes in cultures, reconstructed epidermis in vitro and skin grafted onto nude mice in vivo, of vascular endothelial growth factor (VEGF), an angiogenic factor, and basic fibroblast growth we show that retinoids can inhibit TPA mediated VEGF gene induction at the transcriptional factor (bFGF) in DFSP. By thymidine incorporation, DFSP cells were stimulated to grow 1.5- level. Since retinoids are biologically active either by interacting with the nuclear retinoic acid fold more than normal fibroblasts by exogenous VEGF (33 ng per ml) and 1.45-fold more by receptors (RARs) or by interfering with the AP1 transcription factor, we studied the effect of the exogenous bFGF (10 ng per ml). This cell growth is partially inhibited by antibFGF antibody. By retinoic acid derivative CD 2409, which exhibits strong anti-AP1 activity but does not bind to immunoblotting, DFSP cells produced more VEGF protein than normal fibroblasts and they also the RARs in vitro. The results demonstrate that the inhibition of VEGF expression by retinoids produce VEGF receptor proteins, flt and kdr. We also examined the expressions of VEGF, flt, and only depends on their anti-AP1 activity and does not require gene transactivation via retinoic acid kdr by RT-PCR and DFSP cells expressed flt and kdr more than normal fibroblasts. By response elements (RARE). Since the VEGF promoter contains four potential AP1 binding sites, immunohistochemistry, tumor cells stained positively against anti-VEGF antibody and anti-FGF we used different promoter constructs to identify the functional site responsible for TPA induction antibody. These results may indicate that some angiogenic features may be important in and retinoid inhibition. This site turned out to be localized at position –621 of the 5Ј flanking tumorgenesis, and growth factors including VEGF and bFGF affect the growth of DFSP cells in region of the VEGF gene. paracrine or autocrine manner.

429 430 Coordinate Regulation of EGF-Related Growth Factor Expression and EGF Receptor Activation An Analysis of the Glycosaminoglycan Composition of Keratinocyte CD44, a Candidate Cofactor by 1,25-Dihydroxy-Vitamin D3 in Human Keratinocytes in the Signaling of Heparin-Binding EGF-Like Growth Factors T. Kobayashi and M. Pittelkow . Piepkorn, P. Hovingh and A. Linker Departments of Dermatology and Biochemistry and Molecular Biology, Mayo Clinic, Roches- University of Of Washington School of Medicine, Seattle, WA. Veterans Affairs Medical Center, ter, Minnesota Salt Lake City, Utah Several endogenous EGF-family growth factors [transforming growth factor-01α (TGF-01α), As a membrane proteoglycan regulated by alternative exon splicing, CD44 isoforms participate in amphiregulin (AR), and heparin-binding EGF-like growth factor (HB-EGF)] have been identified diverse cellular functions, which include binding of matrix molecules and soluble ligands. Indirect in human keratinocytes. These autocrine factors play important roles in cell proliferation and evidence suggests that variant exon V3-spliced CD44 is a candidate cofactor in the signaling of differentiation. We show that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], regulates expression and heparin-binding. Because such a function would likely be mediated via the glycosaminoglycan activity of endogenous EGF-family growth factors and epidermal growth factor receptor (EGFR) chains, we analyzed the heparan and chondroitin sulfates of CD44 isolated from human foreskin in human keratinocytes. 1,25(OH)2D3 markedly inhibits DNA synthesis in a time-dependent keratinocytes. The cells were labeled with 35S-sulfate and 3H-glucosamine, the cell lysates were manner. 1,25(OH)2D3 also suppresses EGFR expression and tyrosine autophosphorylation in a extracted with detergent, and the proteoglycans isolated via anion exchange chromatography. dose-and time-dependent manner, with 48 h-treatment markedly decreasing phosphorylated levels CD44 was immunoprecipitated and analyzed in parallel with pooled non-CD44 proteoglycans by of receptor. The effect of 1,25(OH)2D3 on gene expression of EGF-family growth factors was Sepharose chromatography, alkaline borohydride reduction, and selective enzymatic digestion. examined by northern blot analysis. During autocrine proliferation in subconfluent culture, The data indicated that CD44 isoforms comprise ~5% of total cellular proteoglycans. Compared α 1,25(OH)2D3 induced AR and HB-EGF expression but suppressed TGF-01 . HB-EGF expression with non-CD44 proteoglycans, CD44 had a larger relative mass, with Mr Ͼ 100 kDa, and was was also suppressed at 48 h. By contrast, in confluent stationary culture, 1,25(OH)2D3 induced less sulfated. CD44 was completely susceptible to Mr shift upon –BH4 reduction, indicating HB-EGF expression markedly, whereas TGF-01α, AR and EGFR expression were all suppressed.- essentially all molecules to be substituted with glycosaminoglycans. Yields of heparan sulfate chains The present results show that 1,25(OH)2D3 coordinately regulates gene expression of EGF-family were roughly equal to those of chondroitin sulfates, whereas the latter predominated in the non- growth factors and EGFR as well as EGFR phosphorylation. TGF-01α, AR and HB-EGF CD44 pool. CD44 was also heavily substituted with neutral polysaccharides. Our data indicate a expression can be independently regulated by hormones such as 1,25(OH)2D3 and by the growth distinctive pattern of keratinocyte CD44 glycanation, with a relatively high content of heparan state. The complex suppressive or trophic effects of 1,25(OH)2D3 in epidermis may parallel the sulfate. The latter could mediate the growth factor binding properties of the proteoglycan. distinct regulation of growth factor and receptor expression under subconfluent or confluent culture conditions.

431 432 Chronically Elevated Extracellular Calcium Concentration Increases Phospholipase D Activity in Release of Vascular Endothelial Growth Factor from Human Melanoma Cell Lines is Induced by Primary Mouse Epidermal Keratinocytes Anchorage Independent Culture Conditions but Not by Ultraviolet Radiation X. Zheng, S. Ray and W. B. Bollag Y-L. Park, Y. Shellman, J. Gendall, D. Marr and D. A. Norris Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia Department of Dermatology, University of Colorado HSC, Denver Colorado We and others have previously shown that sustained phospholipase D activation is associated with, Vascular endothelial growth factor (VEGF) is an angiogenic factor which promotes tumor growth. and the product(s) of phospholipase D activity can induce, keratinocyte differentiation. In this VEGF release is enhanced by hypoxia, primary cytokines, growth factor withdrawal, and in some study we examined the effect of raising extracellular calcium concentration, a manipulation known systems, by irradiation or by mutational activation of ras signaling. The purpose of this project to induce differentiation, on phospholipase D activity in primary mouse epidermal keratinocytes. was to determine whether VEGF release from human melanoma cell lines was increased by Activity was determined in the presence of small amounts of ethanol by monitoring the formation activating ras mutations or by ultraviolet radiation, and whether dominant negative p53 altered of the novel phospholipid, phosphatidylethanol, a unique marker of phospholipase D activity. In baseline or UV-induced VEGF release. VEGF release from cultured human melanoma cell lines cells labeled with [3H]oleic acid, we found that raising medium calcium levels to 1 mM had no was measured by ELISA (R&D Systems) in media removed from cultures 24 h after irradiation effect on [3H]phosphatidylethanol levels acutely (within 30 min) but increased the formation of with different doses of UVR from a Solar Simulator. VEGF release in monolayer anchorage- [3H]phosphatidylethanol after 24 h to 5.0 Ϯ 0.3-fold over the control value. Phospholipase D is dependent culture and in three dimensional spheroid anchorage-independent culture was also known to use alcohols in addition to ethanol as substrates. We have found that in vitro phospholipase compared in the follow cell lines: WM35 (RGP melanoma) plasmid control, WN35mnras (mutant D can utilize glycerol with a similar efﬁciency to ethanol. In addition, the resultant product, activated ras), WM35dnp53 (dominant negative p53), WM1617 (metastatic melanoma) and a phosphatidylglycerol, has been implicated in cell signaling. Therefore, we examined the effect of plasmid control. In monolayer cultures, ultraviolet radiation from 0 to 100 mJ per cm2 showed a 24-h exposure to elevated extracellular calcium concentration on the subsequent incorporation progressive decrease in VEGF detected in media at 24 h in all cell lines tested. Spheroid culture of [3H]glycerol into phosphatidylglycerol. We found that raising medium calcium levels induced conditions augmented VEGF release in WM35 plasmid control (P ϭ 0.02) and especially in the a large increase in the formation of this phospholipid. We speculate that the production of WM35innias cell line (Increase from 23 ϩ 10.6 to 70 ϩ 18, P ϭ 0.00014); neither the phosphatidylglycerol by phospholipase D may represent a novel signaling pathway by which some WM35dnp53 nor the WM1617 showed signiﬁcant increases in VEGF release in spheroid culture extracellular signals may regulate keratinocyte differentiation. conditions. These results show that VEGF release in human melanomas is not stimulated in the short term by ultraviolet radiation, but that anchorage independent conditions can stimulate VEGF release in some cell lines, especially in the presence of activating ras mutations. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 595

433 434 Stem Cell Factor and Erythropoietin Are Important Factors for Human Skin Mast Cell Growth Ceramides Suppress the Constitutional Activity of Mitogen Activated Protein Kinases, But Do I. Chan, Y. Zhu and M. T. Not Affect Their Activation by Extracellular Signals RPSLMC, Chicago, Illinois T. Kaneko, I. M. Freedberg and M. Blumenberg Stem cell factor (SCF) and IL-3 are important for mast cell (MC) development from bone marrow The Ronald O. Perelman Department of Dermatology, NYU Medical School, New York, precursors; however, little is known about factors that are critical for MC growth in skin. We first New York investigated the effect of SCF (10, 30 and 100 ng per ml) on neonatal foreskin-derived MCs over Three families of mitogen activated kinases, MAPKs, have been characterized in mammalian cells, 2 wk. Thirty ng per ml of SCF was the most effective stimulus for MC growth resulting in nearly ERKs, JNKs and SAPK/p38. Each is activated by a different set of extracellular signals and a 4-fold increase in MCs by day 3 which declined to original culture number by day 14. The phosphorylates different sets of substrates. Ceramide, the common backbone of sphingolipids, can addition of IL-3 (50 ng per ml) or IL-6 (30 ng per ml) to SCF failed to increase the number of act as a second messenger in keratinocytes. Therefore, we examined the effects of ceramide on MCs over 2 wk when compared to SCF alone. IL-4 and IL-10 suppressed the effect of SCF by the activity of MAPKs, using cell permeable C2-ceramide and antibodies specific for the activated nearly 50% within 7 d. Erythropoietin (EPO) has been reported to bind to KIT, the receptor for forms of MAPKs. We found that the ceramide suppressed the constitutive, low level activity of SCF, and thus it was examined for its effects on skin MC cultures. Within 3 d, EPO induced a all three MAPK families in a dose and time dependent way. The constitutive activity of the kinases 3-fold increase in MCs which declined to original starting values by 2 wk. EPO added to SCF that phosphorylate MAPKs was also suppressed. However, the constitutive activity of receptors failed to induce further increases in MCs. To understand the mechanism of action of SCF, skin that initiate MAPK activation, e.g. EGFR, was unaffected by ceramide. Therefore ceramide affects MCs were labeled with BrdU and counter-stained with MC-specific avidin. None of the avidin targets between the receptors and MAPK-kinases. Importantly, ceramide had no effect on the ϩ MCs were labeled with BrdU indicating that SCF did not induce MC proliferation. We activation of ERKs, JNKs and SAPK by EGF, UV light and osmotic shock, respectively. Thus, conclude that SCF increases MCs in culture by promoting their maturation from skin precursors the role of ceramides in MAPK-dependent signal transduction seems to be to attenuate the rather than by proliferation. Like those in peripheral blood, skin MC precursors develop into constitutive level, perhaps allowing a more dramatic fold-induction in response to extracellular mature cells through stimulation by SCF and EPO; however, unlike peripheral blood cells, skin signals. MC precursors are unresponsive to IL-6 and IL-3 and are negatively affected by IL-4 and IL-10. While other unknown factors may be critical for the long-term survival of MCs in culture, it appears that SCF and EPO are important for the maturation of MCs in human skin

435 436 EGF Receptor Signaling Modulates Vitamin A Metabolism in Cultures of Normal and Malig- Enhanced Keratinocyte Spreading on Fibronectin and Elevated Focal Adhesion Kinase (FAK) nant Keratinocytes Levels in Uninvolved Psoriatic Keratinocytes V. Jurukovski* and M. Simon*†‡ G. Chen, T. McCormick, C. Hammerberg, Y. Yoshida and K. Cooper *Department of Oral Biology and Pathology, School of Dental Medicine; Department of Department of Dermatology, Case Western Reserve University and University Hospitals of Dermatology, School of Medicine; †Living Skin Bank, ‡University Hospital, State University of Cleveland, Cleveland, Ohio; Department of Dermatology, University of Michigan, Ann Arbor, New York at Stony Brook, Stony Brook, New York Michigan; and VA Medical Center, Cleveland, Ohio The effect of epidermal growth factor (EGF) on vitamin A metabolism in cultures of normal Abnormal fibronectin signaling through altered integrin expression in combination with soluble human keratinocytes (NHK) and of squamous cell carcinoma (cell lines SCC12b and SCC13) was T cell cytokines may regulate keratinocyte hyperproliferation in psoriasis. Fresh keratinocytes, evaluated. Cells grown in the presence of EGF showed an increase in the accumulation of retinyl obtained from keratomes of uninvolved areas of psoriatic volunteers and from healthy volunteers, esters. Assessment of retinol esterification and retinyl ester utilization (hydrolysis), in cell were incubated on either fibronectin or collagen-laminin mixtures and within one hour homogenates and in cell cultures, revealed that only the enzyme(s) involved in retinyl ester exhibited differential adhesion and spreading. Basal keratinocytes (K1/K10–) from uninvolved hydrolysis were affected. When grown in the absence of EGF, SCC cultures utilized ~40% of areas demonstrated a significantly higher percentage of spreading cells on fibronectin coated plates their retinyl esters in 24 h. NHK utilized ~14% in the same time period. When grown with EGF as compared to keratinocytes isolated from normal skin (52.4% Ϯ 7.5% vs 15.9% Ϯ 3.2%, n ϭ (10 ng per ml), SCC cultures utilized ~20% and NHK utilized ~8%. Differences between SCC 12, P ϭ 0.0002). However, no differences were observed on collagen-laminin coated plates. and NHK utilization rates are due to differences in their capacities to re-esterify retinol; unlike Keratinocytes obtained from psoriatic patients also exhibited a higher basal level of 01α5, 01αv the SCC lines, NHK contains high levels of LRAT (lecithin: retinol acyl transferase). The effect and 01β1 integrin levels when compared to keratinocytes obtained from healthy volunteers. To of EGF was blocked by an EGF receptor neutralizing antibody, an EGF receptor tyrosine kinase address the potential signaling cascades that may respond to integrin changes in psoriatic inhibitor (Tyrphostin51), and a specific inhibitor of MEK kinase influencing the mitogen activated keratinocytes, focal adhesion kinase (FAK) levels were also assessed by immunohistochemistry. protein kinase (MAPK) cascade (PD98059). The effect of EGF on the retinyl ester utilization was The percentage of FAK positive keratinocytes from psoriatic uninvolved areas was greater than also shown to require both transcription and translation. The data suggest that signaling from the the percentage from healthy volunteers after one hour incubation on fibronectin (43.7% Ϯ 8.4% EGF receptor through the MAPK cascade controls the expression and/or activity of retinyl vs 6.6% Ϯ 2.6%, n ϭ 3, P ϭ 0.006). Additionally, FAK activation levels were also assessed on ester hydrolase(s). immunoprecipitated FAK followed by western blot analysis of phosphotyrosine levels on FAK. We found that FAK isolated from uninvolved psoriatic keratinocytes had 10-fold higher levels of activity than FAK isolated from keratinocytes obtained from healthy volunteers. Thus, the proliferative effect of fibronectin in combination with T cell lymphokines on psoriatic uninvolved basal keratinocyte progenitors may be due to abnormal in vivo integrin-driven FAK activity and downstream signaling. This pathway provides a novel potential intervention target in psoriasis.

437 438 Altered Responses to Growth Factors EGF and bFGF in Senescent Human Epidermal Keratinocytes Glial Cell Line-Derived Neurotrophic Factor (GDNF): Involvement in the Control of Epidermal B. Shi and R. Isseroff Homeostasis and in Hair Growth Department of Dermatology, University of California Davis School of Medicine, Davis, California N. V. Botchkareva, V. A. Botchkarev, P. Welker, H. Enomoto, * M. Airaksinen,‡ W. Roth,† C. The proliferative responses of cells to mitogens decrease with aging. We have investigated the Peters,† J. Milbrandt* and R. Paus age-related alterations in the responses to growth factors (GF) in keratinocytes derived from Departments of Dermatology, Charite´, Humboldt University, Berlin, Germany; *Pathology, neonatal foreskin. Keratinocytes were senesced by continued passage. After about 15 passages, Washington University School of Medicine, St. Louis, Missouri; †Hematology & Oncology, cells largely lost the capacity of proliferation. Cells of early (3–5) passage (young) and late (14–16) Albert-Ludwig University, Freiburg, and ‡Inst of Biotechnology, University of Helsinki passage (old) were used for this study. Mobility shift assay demonstrated that DNA binding activity GDNF, a distant member of the TGF-01β superfamily, is critically important for kidney and of AP-1, a major transcription factor for cell proliferation, remained at low levels in both young nervous system development. However, the role of GDNF and its receptor, GFR01α-1, in skin and old keratinocytes maintained in a GF-free medium for 24–48 h. Following stimulation with and hair biology is unknown. Here, we have studied the steady-state levels of GDNF protein EGF or bFGF, AP-1 DNA binding activity in young cells increased by 1–3-fold in a dose- content (ELISA), GDNF and GFR01α-1 gene expression (RT-PCR, in situ hybridization), as dependent manner. These GFs induced little or slight increase in AP-1 binding activity in the old well as the immunoreactivity (IR) for GFR01α-1 in back skin of C57BL/6 mice. GDNF and cells. The MAP kinase inhibitor PD 98059 (10 M) or the tyrosine kinase inhibitor genistein (10 GFR01α-1 expression turn out to be strikingly hair cycle-dependent, and was restricted to defined M) significantly reduced GF-induced AP-1 DNA binding activity in young cells, while PKC epithelial skin and hair follicle (HF) compartments. Maximal steady-state levels of GDNF protein inhibitor GF 109203X (0.5 M) partially inhibited the GF-induced AP-1 activity. Western blot were found in telogen skin (15 pg per mg). Depilation-induced anagen development was demonstrated that EGF induced the translocation of PKC a from cytoplasm into membranes in accompanied by a significant decline of GDNF protein levels in anagen III–IV (P Ͻ 0.05), young cells but not old cells. Application of the growth factors to the young cells increased the followed by an increase during the anagen VI-catagen transformation. GFR01α-1 gene expression expression and phosphorylation of Fos proteins Fra-1 and Fra-2, while the levels of Fra-1 and was maximal in telogen and late anagen, and significantly declined during early anagen (P Ͻ Fra-2 remained lower in the old cells. Our study suggests that the signal transduction mediated 0.05). Basal epidermal keratinocytes (KC) showed GDNF mRNA and GFR01α-1-IR, while by MAP kinase and PKC pathways is altered in senescent human keratinocytes, and these suprabasal epidermal KC displayed GFR01α-1-IR during the entire hair cycle. In telogen hair alterations may be related to the decreased AP-1 transcription activity in old keratinocytes. follicles (HF), GDNF mRNA and GFR01α-1-IR were found in the outer root sheath (ORS). In anagen IV–VI, GDNF mRNA was observed in hair matrix and proximal ORS, while GFR01α- 1-IR was seen in the inner root sheath (IRS). During catagen, we found GDNF mRNA expression on KC of the secondary germ and proximal ORS, while GFR01α-1-IR was present in ORS and the remnants of the IRS. In organ cultured mouse skin, GDNF administration significantly inhibited epidermal proliferation and thickness, retarded the development of anagen hair follicles, and inhibited hair follicle regression (P Ͻ 0.05). Compared to wild type controls, GFR01α-1 knockout mice (ϩ/–) showed a significant (P Ͻ 0.05) acceleration of catagen. Taken together, this suggests important, previously unrecognized roles for GDNF/GFR01α-1 signalling in skin biology, specifically, in the control of epidermal homeostasis and hair growth. 596 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

439 440 Rapid Induction of TNP-Specific Cytolytic CD8ϩ T cells In Vivo by i.c. Injections of TNP- Contact Hypersensitivity (CHS) Induction by Class I or Class II Associated TNP-Modified Modified Dendritic Cells Peptides is Dependent on the Route of Administration S. Martin and J. Simon M. B. Lappin, J. Kohler,* V. D. Delattre, S. Martin, H. U. Weltzien,* T. Preckel* and J. C. Simon Clinical Research Group, Department of Dermatology, University of Freiburg, Germany Department of Dermatology, *MPI, Freiburg, Germany Contact hypersensitivity (CHS) induced by haptens like trinitrophenyl (TNP) is a T cell mediated The identity of the effector T cell population involved in CHS is still questionable with evidence hypersensitivity reaction. The antigenic determinants recognized by TNP-specific T cells from promoting both CD4 or CD8 T cells as the main effectors. Previous experimental studies have C57BL/6 mice have been defined as TNP-modified, MHC-associated peptides. The effector relied on the in depletion of T cell subsets using antibody, or the use of knock-out mice with populations involved in CHS are still a matter of debate although different experimental approaches deficiencies in either CD4 or CD8 T cell mediated immunity. However, to address the role of indicate that both hapten-specific CD4ϩ or CD8ϩ T cells can induce CHS. We have used bone the class I and class II-mediated pathways of T cell activation in CHS responses in mice with an marrow-derived dendritic cells chemically modified with TNP to sensitize C57BL/6 mice for intact immune system, we utilised various TNP-derivatized peptides, which bind specifically with CHS. After TNP challenge on the ears, we detect an ear swelling response only in mice immunized H-2Kb (MHC class I) or H-2I-Ab (MHC class II). The subcutaneous (s.c.) injection of MHC with TNP-modified but not with unmodified DC. When cells from draining axillary and maxillary class II-specific, but not of class I-binding, hapten derivatized peptides in incomplete Freunds lymph nodes (LN) of TNP-DC immunized mice are analysed 72 h after ear challenge, we find a adjuvant (IFA) transferred CHS responsiveness. However, when bone-marrow derived DC were significant induction of IFN-01γ producing CD8ϩ effector cells as shown by ELISPOT assays. pulsed with the same peptides and administered s.c. the opposite result was observed, namely that Little TNP-specific IL-4 production was detected. These effector cells were clearly cytolytic the class I binding peptides induced CHS responses, in contrast class II binding peptides did not towards TNP-modified EL-4 cells as shown aftera3drestimulation in vitro with TNP-modified reproducibly transfer CHS responses. syngeneic spleen cells. To our surprise, we also found TNP-specific cytolytic CD8ϩ effector T Hence in unmanipulated mice it seems likely that both the CD8 and CD4 T cell subset cells after restimulation of LN cells from mice which had been immunized with unmodified DC can mediate CHS responsiveness and that the pathway used is dependent on the route of and then challenged by TNP painting on both ears. These results indicate that the hapten TNP antigen presentation. is an unusually strong antigen which can induce TNP-specific T cells within 72 h in vivo. After 3dofin vitro restimulation these T cells can be identified as TNP-specific cytolytic CD8ϩ T cells. We speculate that the strong T cell response is due to a very high precursor frequency of TNP-specific CD8ϩ T cells as seen in preliminary ELISPOT assays.

441 442 CD40 Ligation Augments CTL Mediated Lysis and Induces Apoptosis in Human Malignant When Do Immune Complexes Cause Vasculitis? Melanoma C. Sunderko¨tter,*† F. Scho¨nlau,* S. Merfeld,* D. Nashan,† R. Hallmann‡ and C. Sorg A. v. Leoprechting, P. v. d. Bruggen, * H. L. Pahl,† A. Aruffo‡ and J. C. Simon *Institute of Experimental Dermatology, †Department of Dermatology, University of Mu¨nster, Department of Dermatology, †Experimental Anesthesiology, University of Freiburg, Germany; Mu¨nster; ‡Institute of Experimental Medicine, University of Erlangen *Ludwig Institute for Cancer Research, Brussels, Belgium; and ‡Bristol-Myers Squibb, Princeton Leukocytoclastic vasculitis (LcV) is usually an immune-complex (IC)-mediated disease. However, Here, we report the functional expression of CD40 on human malignant melanomas (MM). circulating IC occur daily in immunocompetent individuals without causing LcV, e.g. during Comparison of tumor specimen from melanocytic nevi (n ϭ 9), primary tumors (n ϭ 13) and chewing. We wondered (i) how IC could lead to vessel damage, (ii) if IC are sufficient for melanoma metastases (n ϭ 10) revealed a gradual downregulation of CD40 surface expression eliciting LcV, and (iii) how our findings comply with therapeutical strategies. Among circulating during tumor progression. CD40 expression was confirmed by northern blot and FACS in seven IC larger IC may more readily lodge or bind to the vessel wall. In order to imitate the effects of human MM cell lines established from immunogenic primary tumors or metastases. By contrast, vascular deposition we fixed large IC (made of equimolar concentrations of HSA and murine 11 cell lines generated from advanced stages were CD40 negative. CD40 expression could be anti-HSA) to nitrocellulose, added PMN (murine and human) and measured degranulation (01β- enhanced in CD40 positive MM by stimulation with IFN-01γ and TNF-01α but not by IL-101β glucuronidase/LDH). Fixed, large IC caused significantly higher degranulation than the same or CD40-triggering. CD40 ligation on MM by CD40 L-transfected murine L-cells or by a soluble concentration of large IC kept in solution (shuttling) or than small IC (made of AgϾϾAb). Thus, CD40 L-fusion protein induced a rapid activation of the transcription factor NF-01ΦB, upregulated vascular fixation of IC might promote LcV by causing release of proteases at the vesse wall. their expression of ICAM-1, MHC class I and class II molecules, and their secretion of IL-6, IL- However, when screening human dermatoses for vascular deposits of Ig we found vascular Ig also 8, TNF-01α and GM-CSF as shown by EMSA, FACS or specific ELISA. Furthermore, CD40 in nonvasculitic dermatoses (e.g. 30% of porphyria cutanea tarda and 10% of lichen planus) as ligation of a HLA-A2ϩ, MelanA/MART1ϩ MM-cell line enhanced its susceptibility to specific well as in uninvolved skin of patients with LcV. Also in mice after injection of large IC, some lysis by a HLA-A2-restricted, MelanA/MART-1 specific CTL clone. Finally, CD40 ligation vessels were positive for Ig, but not for LcV. Thus, vascular deposits of Ig are not sufficient to induced growth inhibition and apoptosis in MM as shown by clonal growth assay, 3H-Thymidine cause LcV. In search for additional mechanisms we injected neutralizing Ab to E-selectin in the incorporation, DNA-gel electrophoresis, TUNEL and nuclear staining with propidium iodide. murine Arthus reaction (Art-r) and in nonvasculitic irritant contact dermatitis (ICD). This did not These results indicate that CD40–CD40 L interactions may play an important role in regulating change ear swelling or the infiltrate in ICD and the Art-r. However, it significantly reduced antitumor immunity and apoptosis in human malignant melanoma. hemorrhage in the Art-r. High doses of dexamethasone did not inhibit the Art-r in vivo, and also did not reduce degranulation in our in vitro system. In contrast, colchicine (10 µg per ml) significantly reduced degranulation of PMN in vitro as well as expression of E-selectin in vivo and, accordingly, also inhibited hemorrhage in vivo.Thus, IC cause vigorous degranulation of PMN when they are large and fixed, but in vivo need to be combined with the expression of E-selectin to cause LcV. Therapies need to tackle these mechanisms to be effective. E-selectin may induce activation of PMN or formation of small spaces between PMN, IC and endothelial cells in which degranulated proteases cannot be spilled away

443 444 Induction of Langhans Type Multinucleated Giant Cells In Vitro by Muramyl Dipeptide and the Anti-01α-fodrin Antibodies in Lupus Erythematosus and Sjo¨gren’s Syndrome Supernatant of Concanavalin A-Stimulated Mononuclear Cells T. Watanabe, T. Tsuchida, N. Kanda, Y. Hayashi and K. Tamaki K. Mizuno, H. Okamoto and T. Horio Department of Dermatology, Tokyo University, Tokyo, Japan; Department of Dermatology, Department of Dermatology, Kansal Medical University, Moriguchi, Osaka, Japan Saitama Medical School, Saitama, Japan; and Department of Pathology, Tokushima University Murarnyl dipeptide (MDP, N-acetylmuramyl-L-alanyl-D-isoglutamine), a synthetic adjuvant, is a School of Dentistry, Tokushima, Japan structural analogue of part of the bacterial peptidoglycan and produces massive epithehoid It has been reported that sera from an NFS/sld mouse model of human Sjo¨gren’s syndrome (SS) granulornas when injected into guinea pigs and rats. Anti-MIDP antibody showed positive staining reacted with a 120-kilodalton-01α-fodrin protein that is thought to play a crucial role in the of multinucleated giant cells (MGC) in sarcoidal lesions. In the present study, we examined the development of exocrinopathy. We investigated the prevalence of anti-01α-fodrin antibody in the tn vitro effect of MDP on the formation of MGC. The supernatant of concanavalin A-stimulated sera from patients with SS and/or lupus erythematosus (LE) and the association of this antibody human peripheral blood mononuclear cells (conditioned medium) generated both Langhans type- with other clinical manifestations. Nine patients with primary SS, 15 patients with SS secondary (LMGC) and foreign body type-MGC (FMGC) on day 1 with a maximal density on day 3. MDP to LE and 44 patients with LE alone were studied. Anti-01α-fodrin antibody was more commonly augmented the frequency of LNIGC from 15.8% to 40.2% in fusion index, while N-acetyhnuramyl- detected in patients with primary (78%, P ϭ 2.5 01ϫ 10–5) and secondary SS (60%, P ϭ 6.2 L-alanyl-L-isoglutamine (L-MDP), an analogue without adjuvant activity, did not affect the 01ϫ 10–5) than in patients with LE alone (7%). When patients with primary and secondary SS generation of LMGC. Either type of MGC was not generated by MDP from immature dendritic were combined and compared to patients with LE alone, the sensitivity of anti-01α-fodrin cells induced by IL-4 and GM-CSF, although a small number of LMGC was produced from antibody was 67%, the speciﬁcity was 93%, the predictive value positive and negative were both macrophages induced by M-CSF. Immunocytochernical staining showed that both types of MGC 84%. The presence of anti-01α-fodrin antibody was associated with pernio, hyperglobulinemia, expressed CD14, CD36, CD54, CD68 but not CD1a. and CD11b. While CD14 and CD54 were rheumatoid factor positivity and the presence of anti-SS-B (La) antibody (P Ͻ 0.01), but not with stained on the cell membrane, other surface markers and angiotensin converting enzyme were annular erythema, photosensitivity, vasculitis or renal disorder. Although anti-01α-fodrin antibody detected in the cytoplasm of MGC. The architecture of microtubulin in LMGC showed an was detected in both SS and LE, it appeared to be more valuable for the diagnosis of SS than organized pattern, while that in FMGC was irregular. Colchicine inhibited the formation of anti-SS-A (Ro) since anti-01α-fodrin was much less prevalent in patients with LE alone. It might LMGC induced by MDP. These data suggest that MDP plays a role in the pathogenesis of be possible to consider that this novel autoantibody is pathophysiologically associated with some granulomatous diseases characterized by LMGC, such as tuberculosis and sarcoidosis. extraglandular manifestations characteristically seen in human SS. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 597

445 446 Impaired Responses of Mononuclear Cells to Staphylococcal Superantigen in Patients with Severe The Effect of Time and Dose on the Phenotypic Expression of Activation Makers (Cd86 and I- Atopic Dermatitis: A Role of T-Cell Apoptosis Ak) on B Cells Isolated from the DLN of Allergen and Irritant Treated Mice H. Asada, T. Yoshino, S. Sano, T. Nakamura, S. Itami and K. Yoshikawa G. Gerberick, L. Cruse, C. Miller and G. Ridder Department of Dermatology, Osaka University Medical School, Osaka, Japan The Procter and Gamble Company, Cincinnati, Ohio Staphylococcus aureus colonization is an almost ubiquitous feature of atopic dermatitis (AD). In We have previously shown that in mice the number of B lymphocytes increases with allergen order to investigate how staphylococcal superantigen is related to atopic dermatitis immunologically, treatment when compared to the irritant and vehicle control. We have also shown that the median we assessed the correlation between clinical disease severity and proliferative response of peripheral intensity of the accessory molecule CD86 (B7-2) increases on B220ϩ B cells at 72 h following blood mononuclear cells (PBMC) to staphylococcal enterotoxin B (SEB) in patients with AD. the final chemical treatment with allergen, but not irritant. The median intensity of the class II PBMC were isolated from 81 blood samples from 55 patients with AD (27 severe, 27 moderate, antigen I-Ak on B220ϩ B cells remained unchanged with any treatments at 72 h. In these studies, and 27 mild cases) and 19 samples from 14 healthy controls without a personal or family history we examined the effect of time and dose on B cell expression of CD86 and I-Ak in mice treated of AD, and evaluated proliferative responses against SEB. PBMC from patients with mild AD with contact allergen, irritant or vehicle. In the time course study mice were treated on the ears showed significantly increased proliferative responses against SEB compared to controls (P Ͻ for three consecutive days and phenotypic analysis of draining lymph node (DLN) cells was made 0.01), however, PBMC from patients with severe AD showed markedly suppressed proliferative at 24–168 h following treatment. The mice in the dose response study were treated on the ears responses (P Ͻ 0.0001). Additionally, longitudinal evaluation of PBMC samples from the same for three consecutive days with increasing concentrations of allergen or irritant and the DLNs patient demonstrated that the proliferative responses were suppressed only in severe clinical excised 72 h after the final chemical treatment. Phenotypic analyses were performed using flow conditions. To examine the mechanism of these altered proliferative responses in AD patients, we cytometry. As expected, in both the time and dose response studies, the majority of B220ϩ B assessed IL-2 levels in the culture supernatants, but observed no significant differences between cells isolated from the allergen, irritant or vehicle treated mice were CD86 and I-Ak positive. healthy controls and AD groups. FACS analysis for APO2.7 antigen (early apoptosis cell marker) Mice treated with the allergen, dinitrochlorobenzene (DNCB), demonstrated an increase in the demonstrated that approximately 60% of SEB-stimulated T cells expressed APO2.7 antigen in median intensity of CD86 on B220ϩ B cells which was dose dependent and peaked at 72 h severe AD cases, in contrast, 5%–20% of T cells expressed APO2.7 after SEB stimulation in the following the final allergen treatment. This increase was also observed on B cells identified with cases of mild AD and healthy control. In summary, we observed a clear correlation between anti-IgG. Interestingly, the median intensity of I-Ak on B220ϩ B cells isolated from DNCB clinical severity of AD and PBMC proliferative responses to SEB. Furthermore, we found that treated mice increased in a dose dependent manner and peaked at 96 h post treatment. Further suppressed proliferation of PBMC from severe AD patients may be secondary to T cell death by study is required to understand the role of B cell activation/proliferation in the pathophysiology apoptosis. These results suggest that SEB plays a pathogenic role in aggravation of AD by inducing of contact allergy and how it can be used to differentiate allergen and irritant responses in the apoptosis in T cells. draining lymph nodes of mice.

447 448 Role of Fractalkine in Cutaneous Diseases Induction of Antigen-Specific Anergy in T Cells After Coculture with Interleukin-10-Treated S. Raychaudhuri, W. Jiang, E. Farber, T. Schall* Human Dendritic Cells: Analysis of Molecular Mechanisms Psoriasis Research Institute, Palo Alto, California and *Chemocentryx, San Carlos, California K. Steinbrink, H. Jonuleit, G. Mu¨ller, T. Tu¨ting, J. Knop and A. Enk Fractalkine, the latest in the series of chemokines is unique in several aspects: (i) expressed in Department of Dermatology, University of Mainz, Germany endothelial cells, (ii) acts as an adhesion molecule, (iii) chemotactic to the T cells. Recently, we demonstrated that human dendritic cells (DC) after a pretreatment with IL-10 In view of these unique properties fractalkine is capable of controlling key regulatory mechanisms induce a state of antigen-specific anergy in various populations of naive and activated CD4ϩ and of cell trafficking at sites of inflammation. Since fractalkine is discovered recently, little is known cytotoxic CD8ϩ T lymphocytes. Furthermore, functional analysis of anergic tyrosinase-(melanoma- about the role of this important molecule in inflammatory diseases. We undertook this study to associated-antigen) specific cytotoxic CD8ϩ T cell lines revealed a failure to lyse a tyrosinase- investigate the role of fractalkine in inflammatory cutaneous diseases. We used a polyclonal expressing melanoma cell line (SKMEL-29). In this study we investigated possible molecular antifractalkine antibody (immunoperoxidase technique) in cryosections, obtained from tissues of mechanisms of anergy induction in our system. FACS analysis revealed an inhibition of the normal skin and selected cutaneous inflammatory diseases (psoriasis, lichen planus, eczema). upregulation of the costimulatory molecules CD40, CD58 and CD86 and the MHC class II Compared to the control skin we found a high expression of fractalkine in the dermal blood molecules on DC pretreated with IL-10. Investigations of the supernatants of IL-10-treated DC vessels of the inflammatory dermatoses. demonstrated a significantly lower production of IL-101β, IL-6, TNF-01α and the Th1-cell-type The most striking finding was that the dermal dendrocytes adjacent to the keratinocytes in psoriasis inducing cytokine IL-12 compared to control cells. Exploration of surface molecules on the tissues expressed high levels of fractalkine. Compared to 186.64 Ϯ 51.69 fractalkine positive anergic T cells demonstrated an increased expression of CTLA-4 and a downregulation of the dermal dendrocytes in per mm2 of the upper dermis of psoriatic tissue, the number of positive activation marker CD44 and CD69 and the IL-2 receptor 01α-chain. Analysis of the supernatants cells in lichen planus, eczema and normal skin were 17.29 Ϯ 12.50, 12.50 Ϯ 6.75 and 5.93 Ϯ of the anergic T cells after restimulation revealed lower amounts of IL-2 and IFN-01γ but no 3.53, respectively. The findings from this study confirms an upregulation of fractalkine at sites of detectable levels of IL-4 or IL-10, indicating no shift to a Tc2 cytokine pattern. Experiments inflammation; thus it is likely that this molecule play a key role in cell trafficking. Increased levels performed in a boyden chamber system to prevent the attachment of DC and T cells verified that of fractalkine in the dermal dendrocytes, which are potent antigen presenting cells (APCs), is a both the direct cell–cell-contact and the existence of soluble mediators are responsible for anergy unique observation. An increased expression of fractalkine at the dermal papillae provides a induction. Addition of IL-2 or a combination of PMA and ionomycin to the anergic T cell plausible explanation for migration and accumulation of T cells at these sites in psoriasis. It is completely reversed the state of anergy, whereas a combination of anti-CD28-, CD2-CD3-mAb tempting to speculate that the expression of fractalkine on an APC will promote migration of T and the cytokine IL-12 overcame the induction of anergy up to 80%. Kinetic studies using cells specifically targated to the APC and thereby facilitate the interactions with the T cells. restimulation experiments with mature DC at various time points after the onset of the coculture demonstrated that the state of anergy was fully established after 36–48 h. Addition of blocking mAb to CTLA-4, a molecule known to transduce negative regulatory signals, prolonged the time interval for induction of anergy. In contrast, stimulation of the CTLA-4 molecule by antibody crosslinking further decreased proliferation of anergic T cells. Collectively, these data suggest that various molecules and soluble mediators are involved in the anergy induction of T cells by coculture with IL-10-treated DC.

449 450 Development of Genetic Immunization with Mature CD83ϩ Dendritic Cells Peripheral Lymph Node Addressins Are Expressed on Skin Endothelial Cells T. Tu¨ ting, H. Jonuleit, G. Mu¨ller, J. Steitz, J. Bru¨ck, A. Giesecke, K. Steinbrink, J. Knop and A. Enk S. Lechleitner, R. Kunstfeld, C. Messeritsch-Fanta, K. Wolff and P. Petzelbauer Department of Dermatology, University of Mainz, Germany Department of Dermatology, Division of General Dermatology, University of Vienna Medical Cellular immune responses directed against melanoma antigens may be stimulated in vitro using School, Austria PBMC-derived human mature, CD83ϩ DC pulsed with synthetic peptides. As an alternative The term ‘‘peripheral node addressins’’ collectively describes a set of L-selectin ligands, which strategy, we are currently exploring the possibility to genetically modify DC to endogenously react with mAb MECA-79. They are constitutively expressed on lymph node endothelium and express antigens wtih recombinant adenovirus because of their capability to transiently express are required for the continuous lymphocyte recirculation into lymphoid tissues. Such peripheral genes in nondividing cells. Mature, CD83ϩ DC were infected with adenovirus encoding the node addressins have been found absent on postcapillary venules of all other organs. However, model antigen green fluoresecent protein resulting in Ͼ80% fluorescent DC with a mature, veiled under conditions of chronic T cell infiltration, most strongly in cutaneous T cell lymphoma, phenotype due to cytoplasmatic expression of EGFP. Two color FACS analysis simulataneously postcapillary venules of the skin acquire the morphology of high endothelial venules and strongly demonstrates strong staining for MHC class I and II, CD58, CD80, CD83, and CD86 on EGFP- react with mAb MECA-79. To explore whether this extralymphatic MECA-79 reactivity denotes expressing DC. Adenoviral transduction of immature DC does not lead to maturation as assessed the de novo expression of functional peripheral node addressins on skin endothelium, we performed by expression of CD83. Adenoviral vectors encoding melanoma-associated antigens such as gp100, immunoprecipitation studies from skin lysates using recombinant human L-selectin Fc-chimeras. TRP-1, and TRP-2 are being constructed which will subsequently be used to effectively load Indeed, we detected a panel of proteins, which bound to L-selectin and reacted with mAb DC with these antigens. In order to guide clinical investigations, we have also developed culture MECA-79. In contrast to lymph node L-selectin ligands, mainly 90–110 kDa proteins, skin L- conditions for the generation of murine bone marrow-derived DC without the help of foreign selectin ligands additionally comprised a yet undefined 220 kDa protein. Moreover, upon careful serum components. Immunization of BALB/C mice with DC transduced with adenovirus serial sectioning of normal skin, we found perifollicular endothelium constitutively reactive with encoding 01β-galactosidase as a model antigen stimulates antigen-specific CTL responses in vivo. mAb MECA-79. After pooling freshly isolated endothelial cells from 10 cm2 normal skin, L- This system permits us now to investigate in a clincally relevant experimental model the induction selectin immunoprecipitated 90–110 kDa proteins. We postulate, first, that the de novo expression of melanoma antigen-specifc cellular immune responses following immunization with murine DC of peripheral node addressins on skin endothelium plays an important role in the maintenance of transduced with adenoviral vectors encoding gp100, TRP-1, and TRP-2, which have recently chronic inflammation and, second, that the constitutive expression of these ligands on perifollicular been identified as tumor rejection antigens in the murine B16 melanoma model. Ultimaltely, endothelium might be responsible for a continuous lymphocyte recirculation into the skin. these studies will provide a basis for the clinical application of a melanoma vaccine consisting of genetically modified DC for the induction of antimelanoma immunity. 598 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

451 452 Identification of Class II-Restricted Melanoma Antigen in a Long-Lived Patient with Melanoma: Mechanisms of Killing and Escape From Apoptosis Induced by Natural Killer (NK) Cells of Implication in Term of Vaccine Strategies Normal and Immortalized Keratinocytes H. Zarour, W. Storkus and J. Kirkwood B. Bonish and B. Nickoloff Melanoma Center, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania Departments of Microbiology, Immunology, and Pathology, Skin Cancer Research Laboratories, Many MHC Class I melanoma antigens recognized by CD8ϩ T cells have been reported but Cardinal Bernardin Cancer Center, Loyola University Medical Center, Chicago, Illinois few MHC Class II-restricted epitopes recognized by CD4ϩ T cells have been so far identified. The role of the innate immune system in normal cutaneous homeostasis or cutaneous oncology These epitopes are of critical interest given recent reports suggesting that CD8ϩ CTL activity is is not well understood. Cutaneous neoplasms have been shown to be infiltrated by NK cells (key observed in vivo only in the presence of CD4ϩ T cell help. Here we have studied CD8ϩ T and components of innate immunity), but their function and interaction with skin constituents is unclear. CD4ϩ T cells responses against autologous melanoma in a long-lived patient with visceral In order to further delineate the role of NK cells in cutaneous immunology, we established an metastatic melanoma. Mixed lymphocyte-tumor cell culture was performed with autologous in vitro experimental system to examine their effects on benign (primary human keratinocytes) and melanoma tumor cells and peripheral blood lymphocyte (PBMC). After 4 wk of in vitro stimulation, an immortal cell line (HaCaTs). The NK cell line NK-92 was used as an effector cell line. NK- strong and specific T cell responses were observed in 4-h 51Cr-release assays against the autologous 92 cells are positive by flow cytometry for surface markers CD18, CD56, CD94 and CD95L, and tumor cells. Of 50 CD8ϩ CTL clones obtained from limiting dilution assays, 11 showed high negative for CD158a and CD158b. Using chromium release assays, the NK cell line NK-92 was and specific lytic activity against the autologous melanoma cell line. Enzyme-linked (ELISPOT) found to effectively kill both the HaCaT cell line and primary human keratinocytes. For assay of these 11 CTL clones revealed seven HLA-A2 restricted, of which five recognized both keratinocytes, killing was dependent on the confluence of the monolayer, with increasing MART-1/Melan-A 27–35 and 26–35 peptides. confluence being highly protective. At a constant target:effector ration of 10:1, percentage lysis ELISPOT assays using CD8ϩ and CD4ϩ T cells isolated directly from PBMC at various points dropped from 19% at 5 01ϫ 104 keratinocytes per well, 10% at 1.5 01ϫ 105 keratinocytes per of disease course were performed. Strong anti-MART-1/Melan-A CD8ϩ T cell responses and well, to only 5% at 5 01ϫ 105 keratinocytes per well. In contrast, the immortal HaCaT cell line immunoreactvity against one potential MART-1/Melan-A Class II-restricted epitope were was not protected by increasing confluence of the monolayer. At a constant target:effector ratio observed. To confirm these data, CD4ϩ T cells that recognized both antigen-presenting cells of 10:1, percentage lysis was 24% at 5 01ϫ 104 HaCaTs per well, 24% at 1.5 01ϫ 105 HaCaTs pulsed with the MART-1/Melan-A Class II-restricted epitope and the autologous tumor cells per well, and 21% at 5 01ϫ 105 HaCaTs per well. Using appropriate blocking reagents, the NK- were successfully generated from PBMC of normal donors and our patient using autologous 92 cells were found to kill by both a perforin mediated and a Fas/FasL mediated pathway. The dendritic cells pulsed with the Class II-restricted MART-1/Melan-A peptide. perforin pathway was most important in the HaCaTs, while the Fas mediated killing was more In conclusion, we have demonstrated and characterized antitumor CD8ϩ and CD4ϩ T cell important in keratinocytes. Overall our data suggest that tumor cells may be more sensitive to responses associated with long survival of metastatic melanoma. One Class-II restricted MART- NK mediated killing than normal keratinocytes. Confluence can protect normal keratinocytes 1/Melan-A peptide has been identified and could be used in future vaccine trials. from apoptosis induced by NK cells. Since NK cells normally encounter keratinocytes in the context of an intact epidermis, and tumors as solid aggregates of tightly clustered tumor cells, these results reinforce the need to use appropriate experimental conditions such that the in vitro results will have biologically relevant and meaningful significance as regards to NK cell function and cutaneous immunohomeostasis.

453 454 CD44 Variant V10 Mediates Hyaluronan-Independent, VCAM-1-Dependent Adhesion of Gene Delivery to Dendritic Cells in Human Skin Lymphocytes to Inflammatory Dermal Endothelium A. Larregina and L. D. Falo, Jr. and S. N. Wagner, C. Wagner, T. Weimann, B. Gunawardena and M. Goos Department of Dermatology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania Department of Dermatology, University of Essen, Essen, Germany Antigen delivery is a critical challenge in the development of antigen-specific immunlztion CD44 represents a heterogeneous family of cell adhesion molecules containing variant isoforms strategies. Optimal antigen presentation is mediated by professional antigen presenting cells (APCs) (CD44v) with tightly regulated expression on lymphocytes. In contrast to the constitutively capable of processing and presenting antigen to T-cells in the context of costimulatory signals expressed standard CD44 molecule (CD44 s), CD44v10 is upregulated upon activation of required for T-cell activation. The development of strategies to deliver antigens directly to antigen peripheral-blood and skin-infiltrating lymphocytes, on primary nodal and cutaneous lymphomas, presenting cells in vivo is a rationale approach to vaccine design. We have previously demonstrated, and on circulating Se´zary’s cells. Whereas the role of CD44 s in localization and extravasation of using murine models, that biolistic cutaneous delivery of naked DNA results in the transfection of lymphocytes has been ascribed to recognition of endothelial HA induced by proinflammatory cutaneous dendritic cells and stimulates potent CTL-inediated immunity. To develop immunization stimuli such as TNF-01α and IL-101β, the role of CD44v10 has been defined not yet. We strategies based on the delivery of antigen-encoding genes to human dendritic cells in vivo,we generated Namalwa cells stably transfected with CD44v10 and CD44 s and observed binding of have utilized a human skin explant model. Our studies demonstrate that human skin explants can these transfectants to TNF-01α and IL1-01β treated human dermal microvascular endothelial cells remain viable and maintain the anatomic architectural features of intact skin for several days. (HMEC). However, preincubation of either cell population with hyaluronidase or Ab MEM-85 Cutaneous dendritic cells in these explants display phenotypic and morphologic characteristics (directed against the HA-binding domain of CD44) did not abolish this interaction indicating a remarkably similar to those of dendnitic cells in human skin in vivo. The accumulation of dendritic HA-independent mechanism of adhesion. Blocking studies with a series of Abs (including anti- cells in the dermis in cords along lymphatic vessels and in the supernatants of explant cultures CD11a, CD18, CD49d, CD54, CD62L, CD62E, CD106, CLA, CD44 s, CD44v10, and isotype- over time is consistent with in vivo dendritic cell migration. To evaluate gene delivery, the gene matched controls) revealed marked reduction of adhesion to HMEC by anti-VCAM-1 Ab. encoding Green Fluorescent Protein was biolistically delivered to freshly excised human skin. Although flow cytometry revealed comparable surface expression levels of VCAM-1-receptor After 48 h, migrating dendritic cells were collected and analyzed. Electronmicroscopy demonstrated VLA-4 on CD44v10ϩ, CD44 sϩ, and CD44– Namalwa cells, CD49d-specific Ab blocked the presence of gold particles in skin-derived dendritic cells. Importantly, colocalization of adhesion of CD44 sϩ and CD44– completely, but not of CD44v10ϩ lymphocytes. Combinations green fluorescence (EGFP expression) in HLA-DR expressing cells (red fluorescence) could be of anti-VLA-4 with -VCAM-1 or -CD44v10 antibodies markedly reduced CD44v10-binding demonstrated in the migratmig cell population, and in resident dendritic cells in the epidermis further, supporting a CD44v10/VCAM-1–dependent interaction. This is the first report on and dermis. Together, these studies suggest that human cutaneous dendritic cells can be transfected differential recognition of EC ligands by CD44 sϩ and CD44vϩ lymphocytes, suggesting an in vivo, and establish the suitability of this system as a model for the development of in vivo human additional mechanism for regulation of lymphocyte adhesion and trafficking. cutaneous genetic immunization strategies. Supported by NIH IR21 A14269701

455 456 CD26 (Di-Peptidyl-Peptidase IV) is Constitutively Expressed by Basal Keratinocytes in Normal IL-11 is an Immune-Modulatory Cytokine which Downregulates IL-12, Type 1 Cytokines, and Skin and is Overexpressed in Psoriatic Keratinocytes Multiple Inflammation-Associated Genes in Patients with Psoriasis M. V. Rivas, M. Ozawa and J. G. Krueger W. Trepicchio, M. Ozawa, IB. Walters, T. Kikuchi, U. Schwertschlag, A. Dorner and J. G. Krueger Investigative Dermatology, Rockefeller University, New York, New York Investigative Dermatology, Rockefeller University, New York, New York and Genetics Institute, Using mRNA differential displays, we identified increased expression of CD26 in lesions of Cambridge, Massachusetts psoriasis vulgaris compared to uninvolved and normal skin controls. CD26 is a T-cell surface Psoriasis is a T-lymphocyte-mediated disease typified by overproduction of IL-12 and pro- molecule that is up-regulated on activated T-cells and functions as an exoprotease. Evidence inflammatory type I cytokines in skin lesions. In vitro and in vivo studies in animal models have suggests that CD26 regulates both T-cell activation and extracellular processing of numerous demonstrated that rhIL-11 downregulates IL-12 and Th1 T-cell responses. Hence, the potential inflammatory cytokines and chemokines. In psoriatic lesions, CD26 mRNA was significantly up- of rhIL-11 to modulate chronic inflammation in lesions of psoriasis vulgaris is being tested using regulated in basal and spinous keratinocytes, and CD26 protein expression paralleled mRNA daily s.c. injections over an 8-wk treatment period. Six patients in the lowest dose cohort (2.5 µg distribution. Surprisingly, abundant CD26 mRNA was also detected in basal keratinocytes of per kg) of a Phase I study have completed treatment. Tissue was obtained from uninvolved and normal skin by in situ hybridization and by immunocytochemistry. Northern analysis showed lesional skin by biopsy prior to treatment with rhIL-11. Lesional skin biopsies were obtained at constitutive expression of the expected 4.2 kb mRNA in all skin substrates with an additional 2.8 weeks 1, 4, and 8 during IL-11 administration and were examined by immunohistochemistry or kb mRNA in psoriatic skin. CD26 expression by normal basal keratinocytes was confirmed by quantitative RT-PCR for T-cell infiltration, production of pro-inflammatory cytokines/mediators, immunohistochemistry with the monoclonal antibody L272 and by in situ enzyme histochemistry and epidermal hyperplasia. Expression of multiple proinflammatory genes was elevated in psoriatic with substrate glycyl-L-proline-4-methoxy-2-naphthylamide. In cultured keratinocytes, CD26 tissue compared to nonlesional skin as determined by RT-PCR. In patients whose biopsy site mRNA was induced by high-dose insulin, bovine pituitary extract, and by gamma-interferon, lesion scores improved with rhIL-11 treatment, expression of 10 disease-related genes including and down-regulated by 1,25 dihydroxycholecalciferol and 12-O-tetradecanoylphorbol-13-acetate. K16, iNOS, IFN01γ, IL-8, IL-12, TNF01α and IL-101β was downregulated, but IL-4 expression Thus, CD26 mRNA appears to be regulated by mitogenic and inflammatory cytokines in human increased. Changes in inflammatory gene expression correlated with reductions in the number of keratinocytes. Hence, keratinocytes could function as direct regulators of cutaneous inflammation immunoreactive K16ϩ, Ki67ϩ, and ICAM-1ϩ keratinocytes. Comparable changes were not via CD26. observed in nonresponding patients. These are the first data which suggest that excessive production of IFN01γ and other pro-inflammatory cytokines can be counter-regulated cytokine administration in a human disease. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 599

457 458 DAB389 IL-2 Induces Apoptosis of Mature Dendritic Cells in Psoriasis Vulgaris Lesions Dendritic Cell Maturation and Subsequent Enhanced T-Cell Stimulation Induced with the Novel B. Holder, L. Austin, J. Carucci and J. Krueger Synthetic Immune Response Modifier R-848 Investigative Dermatology, Cellular Physiology and. Immunology, Rockefeller University, New C. L. Ahonen, S. J. Gibson, R. M. Smith, L. K. Pederson, R. L. Miller, M. A. Tomai and J. York, New York P. Vasilakos Psoriasis is a T-cell mediated disease characterized by activated lymphocytes. Activated T cells 3M Pharmaceuticals, Department of Pharmacology, 3M Center, St. Paul, Minnesota express the IL-2 receptor (R) consisting of the alpha (CD25) and constitutively expressed beta Dendritic cells are the most potent antigen presenting cells. Agents that enhance dendritic cell chain (CD122) as a dimer on the cell surface. DAB389IL-2 is a chimeric toxin which kills IL- maturation can enhance T cell activation and therefore improve the efficiency of vaccines or 2Rϩ cells via apoptosis and is effective in clearing psoriatic lesions. Concurrent with thinning cellular immunotherapy. Previously, we demonstrated that the low molecular weight synthetic (healing) lesions, there is also a reduction in T cell numbers. Mature dendritic cells (DCs), like immune response modifier, R-848 (4-amino-2-ethoxymethyl-a,a-dimethyl-1H-imidazo[4,5-c]qui- activated T cells, express the IL-2(R), both CD25 and CD122, while immature DCs do not noline-1-ethanol), induces IL-12 and IFN-01α secretion from monocytes and macrophages in vivo express CD25. The purpose of this study was to determine if DAB389IL-2 induces apoptosis of and in vitro. Determine if R-848 can induce the maturation of monocyte-derived dendritic cells. DCs both in lesions of treated patients and in DCs derived from normal blood donors. Biopsies Dendritic cell maturation was determined by cell surface expression of maturation markers detected were obtained from patients with chronic plaque psoriasis treated intravenously with DAB389IL- by flow cytometry, cytokine secretion detected by ELISA or bioassay and allogeneic and syngeneic 2. Patients were evaluated clinically and skin biopsies obtained from both lesional and nonlesional T cell activation determined by T cell proliferation and cytokine secretion. Characteristic of areas before and after one treatment. Cryostat sections of skin biopsies were evaluated by dendritic cell maturation, R-848 treatment induces cell surface expression of CD83, and increases immunohisto-chemistry for loss of cell surface markers on immature and mature dendritic cells. cell surface expression of costimulatory molecules CD80 and CD86, as well as CD40 and HLA- A reduction of CD86ϩ and CD83ϩ cells was observed in addition to loss of CD3ϩ cells in DR. Additionally, R-848 induces cytokine (IL-6, IL-12, TNF01α, IFN-01α) and chemokine healing lesions. In biopsies evaluated 8 d after the first DAB389IL-2 treatment, TUNELϩ cells, (IL-8, MIP101α and MCP-1) secretion from dendritic cells. Most significantly, the immune which indicate apoptosis were colabeled with CD83 in the dermis and basal layer of the epidermis response modifier enhances dendritic cell antigen presenting function as measured by increased T of lesional skin. The ability of DAB389IL-2 to induce apoptosis and death in mature DCs, was cell proliferation and T cell cytokine secretion in both allogeneic and autologous T cell systems. then directly evaluated in vitro. Normal human blood derived mature DCs (CD83ϩ) were treated The quantitative T cell responses correlate with the amount of MO-DC CD83, HLA-DR, CD80, with different concentrations of DAB389IL-2 and observed at several time-points after treatment. CD86 and CD40 expression as well as MO-DC cytokine production. These effects are dose With increasing doses of DAB389IL-2, FITC-annexin V binding also increased followed by a loss dependent and are consistently seen with 2 µg per ml R-848 and less marked with 0.4 µg per ml of cells consistent with death by apoptosis. We propose that DAB389IL-2, in addition to killing R-848. The data indicate that the immune response modifier R-848 directly enhances the ability activated T cells, can also kill mature DCs. of human monocyte-derived dendritic cells to activate T cells. Consequently, low molecular weight synthetic molecules such as R-848 and its derivatives may be useful as vaccine adjuvants or as ex vivo stimulators of dendritic cells for cellular immunotherapy.

459 460 Correlation of Patch Test and Lymphocyte Proliferation Responses in Two Patients with a History Calcitonin Gene-Related Peptide-Immunoreactive Nerves in Prurigo Nodularis – An Exploration of Acute Generalized Exanthematous Pustulosis of Neurogenic Inflammation M. Girardi, J. McNiff, K. Duncan, R. Tigelaar, B. Steimer, S. Imaeda and K. Watsky Y. Liang and O. Johansson Department of Dermatology, Yale University School of Medicine, New Haven, Connecticut Experimental Dermatology Unit, Department of Neuroscience, Karolinska Institutet, Stock- An adverse cutaneous reaction to a systemically administered drug may rarely manifest as acute holm, Sweden generalized exanthematous pustulosis (AGEP). Several reports have documented positive patch The aim of the study is to explore the localization and distribution of calcitonin gene-related test results in patients with a history of AGEP. One has reported histology of the patch test site peptide (CGRP)-immunoreactive (IR) nerve fibers in prurigo nodularis, especially emphasizing to be AGEP-like, while another has reported spongiotic dermatitis. We describe two patients with the relationship to mast cells and eosinophils, which all are important contributors to neurogenic a history of AGEP who each demonstrated positive patch test results specific for the inciting drug inflammation. The exact localization of CGRP in nerve fibers of prurigo nodularis lesional skin (1% in petrolatum): Patient #1 to the antibiotic metronidazole, and Patient #2 to the calcium has been clarified by an ultrastructural immunogold labeling technique; and the relationship of channel-blocker diltiazem. Histologic examination of biopsy specimens taken from the positive CGRP-IR nerve fibers to tryptase-IR mast cells and eosinophil cationic protein (ECP)-IR patch test sites of these patients revealed epidermal spongiosis with a superficial perivascular and eosinophils was also investigated by immunofluorescence double-labeling. The ultrastructural study focally interstitial infiltrate, rather than features more typical of AGEP (i.e. spongiform pustulation has demonstrated that CGRP immunoreactivity is increased in the dense-core vesicles in the and subcomeal collections of neutrophils with papillary edema and a polymorphous perivascular axons of prurigo nodularis lesional skin; the axons which contain CGRP are, in addition, infiltrate). Peripheral blood leukocytes were isolated from both patients and assayed in vitro by enlarged and have more dense-core vesicles than the axons which do not contain CGRP. The measuring T cell proliferative responses to purified drug. Responses were consistent with the immunofluorescence investigation demonstrated that tryptase-containing mast cells and ECP- clinical findings: Patient #1 demonstrated a statistically significant (p 01ഛ 0.05) increase in containing eosinophils are also significantly increased in the lesional skin. The results indicate that leukocyte proliferative response to metronidazole (1 ptg per ml), when compared to Patient #2 certain neurons increasingly express CGRP, and this may dynamically result in a neurogenic and control leukocyte responses to the same stimulator. In addition, Patient #2 showed a significant inflammation in the lesional skin, fulfilled through vasodilatation and recruitment of inflammatory (p 01ഛ 0.05) increase in proliferative response to diltiazem (1 µg per ml) relative to normal cells, e.g. eosinophils and mast cells. control leukocytes. That both of our patients showed specific patch test reactivity to their respective AGEP inciting drugs, and that these findings were consistent with the observed proliferative responses in vitro, supports the notion that AGEP occurs via a drug-specific, T cell-mediated process. Since AGEP clinically resembles pustular psoriasis, and since evidence exists that psoriasis is a T cell driven disorder, AGEP and psoriasis may share some pathogenic events.

461 462 Diverse Effects of CGRP and NO on Immunologic and Nonimmunologic Reactions in Human Melanoma Cells and Melanocytes Produce Interferon (IFN)-01α and IFN-01β Human Skin H. Fujisawa, B. Wang* and F. Otsuka J. Wallengren Department of Dermatology, Institute of Clinical Medicine, University of Tsukuba, Japan; Department of Dermatology, Lund University Hospital, Lund, Sweden *Sunnybrook Health Science Centre, Division of Dermatology, University of Toronto, Canada There is much evidence of an interaction between the nervous and the immune systems. The Interferon (IFN)-01α and IFN-01β are utilized in melanoma therapy. However, whether or not aim of this study was to investigate the role of two putative neurotransmitters, calcitonin gene- melanocytes and melanoma cells produce IFN-01α or IFN-01β has been unclear. In this study, related peptide (CGRP) and nitric oxide (NO), in the modulation of immediate and delayed, we demonstrate that human melanocytes, melanoma cell lines such as HMV-1, MMAc, and immunologic and nonimmunologic reactions in human skin. CGRP, 13 pmol and 39 pmol, VMRC-MELG cells form IFN-01α or IFN-01β. We found that culture supernatants from CGRP antagonist-CGRP(8–37), caimed to act as receptor antagonist, 0.05 nmol and 0.5 nmol, unstimulated human melanocytes and melanoma cell lines did not contain detectable amounts of nitroglycerin (a NO donnor), 0.1 µg, and a nitric oxide synthase inhibitor (NG-nitro-L-arginine) IFN-01α or IFN-01β. IFN-01α was detected in the supernatants of HMV-1 cells stimulated with L-NAME, 0.1 µg, were injected intracutaneously, prior to provacation tests. CGRP, 13 pmol, the potent IFN inducer polyinosinic:polycytidylic acid (poly I:C). Supernatants from human inhibited allergic contact dermatitis (P ϭ 0.063). CGRP(8–37) exerted dual effects on allergic melanocytes, MMAc and VMRC-MELG cells activated with poly I:C showed the presence of contact dermatitis causing potentiation in a dose of 0.5 nmol (P ϭ 0.004) and inhibition in a dose IFN-01β. The level of IFN-01α or IFN-01β mRNA was also upregulated in IFN-01α or IFN- of 0.05 nmol (P ϭ 0.012). L-NAME inhibited irritant contact dermatitis (P ϭ 0.020) but b protein producing cells. The effects of IFN-a and IFN-01β on the proliferation of the melanoma augmented delayed immunologic reaction from tuberculin (P ϭ 0.06) and immediate immunologic cell lines were also investigated. IFN-01α and IFN-01β (Ͻ1000 IU per ml) suppress the reaction from birch (P ϭ 0.016). None of the compunds inﬂuenced UVB induced dermatitis.The proliferation of all types of melanoma cells. The suppressive effects of IFN on the proliferation of results suggest that CGRP and NO participate in different mechanisms in the defence system. melanoma cells are likely to be a common property in melanoma cell lines suggesting that CGRP and NO can thus alter in vivo immune responses and their analogues or antagonists may melanoma cell cannot acquire resistance against IFN-01α and IFN-01β in vivo. The cytokine gene perhaps be used as immunomodulatory therapeutic agents. transfer of IFN-01α and IFN-01β to non-IFN producing melanoma cells or the stimulation of IFN production from melanoma cells with IFN producing ability could be useful in the treatment of melanoma patients. 600 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

463 464 Differential Profiles of Cytokines and Adhesion Molecules of Lymph Node and Splenic Dendritic HPLC Determination of Metabolites of the Inflammatory Key Enzymes Using a Diode-Array- Cells in the Mice with Down-regulation of Contact Hypersensitivity UV-Detector and a Light-Scattering-Detector in Series T. Kim,* H. Hwang, S. Choi and C. Kim A. Schepky, R. Siegner and W. Diembeck Department of Dermatology, Kangnam St. Mary’s Hospital, the Catholic University of Korea, Department of Biocompatibility/Biochemistry, Paul-Gerson-Unna Skin Research Center, Seocho-gu, Seoul, Korea Beiersdorf AG, Hamburg, Germany Contact hypersensitivity is a T cell-mediated immune response initiated by dendritic cells (DC) An approach is presented to model the in vivo situation of the inflammation cascade key enzymes’ and the development of specific T cells during the response are affected by cytokines and adhesion metabolisms (phospholipase A2 [3.1.1.4], prostaglandin synthase [EC 1.14.99.1], and lipoxygenases molecules released by DC. In the present study, the mice were induced suppression of contact [EC 1.13.11.12]) simultaneously in an in vitro test assay. Their activities generate some arachidonic hypersensitivity by i.v. administration of 5 mg TNBSO3 3 h after sensitization (desensitization acid metabolites which act as potent mediators of a variety of inflammations, including skin group) or 7 d before sensitization (tolerance group). DC of lymph node were isolated by density diseases as well. Detection of phospholipids (up to 50 pM) and arachidonic acid (up to 33 pM) is centrifugation, and DC of spleen were isolated by magnetic positive cell separation. The expressions performed with high sensitivity and without radiolabelling using a SEDERE light scattering of cytokines (IL-101α, IL-101β, IL-6, IL-10, IL-12 p35, IL-12 p40, and TNF-01α) and adhesion detector. Thus, in combination with a diode array detector, lipids, prostaglandins, HETEs and molecules (B7-1, B7-2, ICAM-1, LFA-101α, and LFA-101β) of DC were investigated by other metabolites of the inflammation cascade can be determined with high efficiency using a semiquantitative RT-PCR. In DC of lymph node, by comparision with DC of TNCB-immunized reversed phase-high performance liquid chromatograph and on-line detection. group (positive control; PC), IL-101α, IL-6 and TNF-01α mRNA in tolerance group, and IL-6 Furthermore, with this sophisticated analytical method examination of ex vivo samples from human in desensitization group were rarely expressed at day 3 and day 5 after priming. And expression skin should be feasible to study the effect of enzyme inhibitors in appropriate vehicles after topical of IL-12 p35 and p40 mRNA in both groups were slightly decreased. However, IL-10 mRNA application to skin. were transcripted similar to levels observed in PC. In addition, B7-1, B7-2, ICAM-1, and LFA- 101α mRNA showed weak expression at day 5 in DC of both groups compared to DC of PC. In DC of spleen, expression of IL-101α, IL-12 p40 and TNF-01α were down-regulated in desensitization group. However, IL-10 mRNA expression in tolerance group was markedly elevated on day 5, and IL-12 p35 and p40 mRNA levels were slightly increased above levels observed in PC. Desensitization group did not show much differences with PC in the expressions of adhesion molecules, but B7-1 mRNA expression in tolerance group were increased compared to that of PC. These observations suggest that DC of lymph node and spleen have functionally discrete roles and may be differently involved in down-regulation of contact hypersensitivity.

465 466 C1q Inhibits Autoantibody Binding to Calreticulin Human Skin Cells in Vitro Express the Neuroendocrine -Specific Pro-Hormone Convertase 2 D. M. Racila and R. D. Sontheimer Cofactor 7B2 Department of Dermatology, UT South-western Medical Center, Dallas, Texas; and Department T. Fisbeck,* M. Schiller,† H. Kalden,*† T. Brzoska,† T. Schwarz,*† M. Bo¨hm*† and T. Luger*† of Dermatology, University of Iowa College of Medicine, Iowa City, Iowa *Ludwig Boltzmann Institute for Cell Biology and Immunobiology of the Skin and †Department Calreticulin (CR) is a new rheumatic disease-associated autoantigen that is intimately associated of Dermatology, University of Mu¨nster, Germany with the Ro/SS-A ribonucleoprotein. CR autoantibodies are frequently observed in patients with Generation of the biologically active peptide hormones adrenocorticotropin (ACTH) and 01α- photosensitive forms of lupus erythematosus (LE). CR has been shown to be highly homologous melanocyte-stimulating hormone (01α-MSH) requires the proteolytic activity of a subset of to cC1q-R, the cell surface receptor that binds the collagenous domain of the first component of enzymes known as the prohormone convertases (PCs). Processing of proopiomelanocortin (POMC) complement, C1q. C1q has also been shown to directly bind to CR. We therefore asked whether is tissue-specific and performed by PC1 (yielding ACTH and 01α-lipotropin), or by a combination the binding of C1q to CR might interfere with the binding of CR autoantibody to CR. Full- of PC1 and PC2 (yielding 01α-MSH and 01β-endorphin). In contrast to the well-described length recombinant human CR derived from an E. coli fusion protein was used as antigen in a expression of these enzymes in the central nervous system, little is known about its expression in direct enzyme-linked immunosorbent assay (ELISA). CR autoantibody-containing sera were human skin cells. In light of the postulated concept of the skin as an extraneural and locoregional assayed before and afinterfere with the binding of CR autoantibody to CR. Full-length recombinant source for POMC-derived peptides, we set out to examine the expression of PC1, PC2, and the human CR derived from an E. coli fusion protein was used as antigen in a direct enzyme-linked neuroendocrine-specific polypeptide 7B2, whose expression and binding to PC2 determines immunosorbent assay (ELISA). CR autoantibody-containing sera were assayed before angher in maturation and enzyme activity of the latter. Due to the lack of commercially available antibodies sera depleted of C1q by all three methods compared to unmodified sera. The addition of purified against these peptides, we determined their expression in various skin types by RT-PCR. RT- C1q to C1q-depleted sera resulted in ELISA OD values similar to those of unmodified sera. These PCR with primers against PC-1 confirmed specific transcripts in normal human keratinocytes, results suggest that C1q levels present in human serum can inhibit the binding of CR autoantibody microvascular endothelial cells, dermal fibroblasts and melanocytes. Although we could not detect to CR. The failure of C1q to mask CR autoepitopes in individuals with genetic deficiency of RNA transcripts for PC2, 7B2 was expressed at the RNA level in freshly prepared human C1q could contribute to the high rate of photosensitive LE that occurs in such individuals. monocytes, keratinocytes, microvascular endothelial cells, dermal fibroblasts and melanocytes as well as a in panel of immortalized cell lines including HaCaT keratinocytes, the monocytic cell line U937, Fs4 fibroblasts and two melanoma cell lines (WM35 and WM9). Since we have previously shown that PC1 expression in keratinocytes can be induced by UVB, regulation of 01α-MSH generation at the level of POMC processing appears to be an important step by which pigmentary, immunomodulatory and proliferative signals are coordinated in the skin.

467 468 STAT6 Plays a Critical Role in Contact Sensitivity (Cs) The Expression of CC Chemokine Receptor 3 (CCR-3) is Detectable in Normal Human Skin H. Yokozeki, M. Ghoreishi, K. Takavama and K. Nishioka and is Enhanced in Inflammatory Skin Diseases: RANTES, But Not Eotaxin, IL-4 or Interferon- Department of Dermatology, School of Medicine, Tokyo Medical and Dental University, 01γ, Up-Regulates CCR-3 Expression on Cultured Human Keratinocytes Tokyo, Japan M. Wakugawa, K. Nakamura, M. Akatsuka, H. Kawasaki, K. Tamaki and M. Furue STAT6 play a central role in exerting not only IL-4 but also IL-13 mediated biological responses, Department of Dermatology, University of Tokyo, Tokyo Japan; Research and Development however it is not clear that the role of Th 2 type ctyokines in the sensitivity (CS). The purpose Laboratories, Maruho Co. Ltd, Kyoto, Japan; Department of Clinical Immunology and AIDS of these studies was to analyze the functional contribution of Th 2 cytokines in CS using the Research, Institute of Medical Science, University of Tokyo, Tokyo Japan; and Department of STAT6 deficient mice. STAT6 deficient (STAT6–/–) mice and wild type (WT) mice (C571BL/ Dermatology, Kyushu University, Fukuoka Japan 6) were contact sensitized with 5% TNCB, 0.5% DNFB, or 5% Oxa. The maximal ear swelling CC chemokines are known to participate in chemotaxis of infiltrating cells in inflammatory was detected at 24 h for the WT mice, however the ear-swelling response was significantly and diseases. However, the role of CC chemokine receptor 3 (CCR-3), one of the receptors for dramatically reduced after 24 h and a peak response was detected at 72 h in STAT6–/–mice. In RANTES and MCP-3 and the only receptor for eotaxin, has not been well elucidated in skin contrast, our studies showcd that STAT6–/– mice did not exhibit a reduced irritation response inflammation. The expression of CCR-3 in normal and several diseased skin was examined by nor delayed type hypersensitivity induced by sheep RBC. The reduced number of Langerhans immunohistochemical procedure. In normal skin, CCR-3 was expressed on keratinocytes (KCs), cells but not TCR01γδ cells observed in epidermal sheets prepared from STAT6–/– mice was not dermal vascular endothelial cells (DVECs) and fibroblasts. In diseased skin, CCR-3 expression responsible for the decrease ear swelling in STAT6–/– mice, since the allostimulatory capacity of was also detectable in an augmented fashion in these skin components and dermal infiltrating cells. enriched Langerhans cells prepared from STAT6ϩ/ϩ and STAT6–/– mice was comparable in vitro. Especially augmented CCR-3 expression was observed on fibroblasts, eosinophils and CD4ϩ T The phenotype analysis of effector T cells prepared from lymph nodes in TNCB contact sensitized cells. Next, we examined the CCR-3 expression on cultured normal human KCs. These KCs WT mice and in STAT6–/– mice revealed that Thy 1ϩ TCR01γδ ϩ IL-4R ϩ V01γ3 ϩ CD3 expressed CCR-3 at protein and mRNA levels. FACS analysis revealed that cultured human KCs ϩ T cells was present in WT mice (12%) but absent in STAT6–/– mice. An enhanced ear expressed CCR-3 and this was up-regulated by RANTES, but not by eotaxin, IL-4 or Interferon- response was ohserved 24 h after challenge when TCR01γδϩ T cells from TNCB sensitized WT 01γ. These results indicate that CCR-3 is constitutively expressed on KCs, DVECs and fibroblasts mice were given to TNCB sensitized STAT6–/– recipient mice. Our data suggest that STAT6 and is up-regulated in inflammatory skin diseases. Furthermore, the expression of CCR-3 on KCs signal is essential at the effector stage of the CH. is selectively up-regulated by RANTES. Thus, our data indicate that CCR-3 and RANTES interaction is involved in the process of skin inflammation. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 601

469 470 Prevalence and Antigen Specificity of Antihistone Antibodies in Patients with Polymyositis/ Noninvasive Method for Assessing Inflammatory Changes in Chemically Treated Human Skin Dermatomyositis M. A. Perkins, M. A. Osterhues and M. K. Robinson M. Kubo, H. Ihn, N. Yazawa, S. Sato, K. Kikuchi and K. Tamaki The Procter & Gamble Co., Cincinnati, Ohio Department of Dermatology, Faculty of Medicine, University of Tokyo, Tokyo, Japan; and We previously developed a noninvasive method to assess inflammatory changes in human skin. Department of Dermatology, Faculty of Medicine, Kanazawa University, Kanazawa, Japan Our approach is based on simple tape adsorption (Sebutape) of molecular mediators of skin Antihistone antibodies (AHA) have been detected in the sera of patients with various autoimmune irritation (e.g. cytokines) from the skin surface. The Sebutape method quantitatively extracts diseases. However, the existence of AHA in patients with polymyositis/dermatomyositis (PM/ detectable levels of constitutive (e.g. Interleukin-101α, IL-1 receptor antagonist) and induced (e.g. DM) has not been reported. We found AHA in 8 (17%) of 46 sera from patients with PM/DM IL-8) cytokines from skin at various body sites. This procedure was used to investigate baseline by enzyme-linked immunosorbent assay (ELISA). One serum was positive for both IgG AHA condition and changes in skin due to chemical or physical insult and existing dermatitis. In infant and IgM AHA. Six sera were positive only for IgG AHA. One serum was positive only for IgM product use tests significant increases in IL-1, IL-1ra, and IL-8 were found in the diaper rash vs. AHA. An indirect immunofluorescent analysis using HEp-2 cells as the substrate showed that all control skin sites with a positive correlation between IL-1ra levels and diaper rash severity. Also sera positive for AHA produced homogeneous nuclear fluorescence. These immunofluorescence a correlation was found between the degree of rash severity and the levels of IL-8. The current patterns disappeared after absorption of antihistone activity with total histones. An immunoblotting studies were designed to further characterize the Sebutape sampling method and explore its’ analysis demonstrated that the AHA were predominantly directed against histone H1 in all seven application for assessment of chemically damaged skin. To evaluate the invasiveness of the Sebutape sera with IgG AHA. Weak reactivity with H2B and H4 were also found in three sera from the procedure and assess cytokine levels recovered in consecutive samples from the same skin site, 30 patients with PM/DM. Sera from two patients with PM/DM displayed anti-H2 A and H3 activity. samples were collected from normal untreated forearm. To determine the methods utility for One of the two sera showed IgM AHA in the ELISA reacted with H1, H2 A, H2B, H3 and H4, assessment of chemically irritated skin, the cytokine responses of forearm skin, patched with either while the other serum reacted with no fractions of total histones. The activity of AHA disappeared 20% SLS or water vehicle for 2 h, were compared to a naı¨ve (untreated) control site. After in immunoblotting after absorption with total histones. All of the patients with AHA were free treatment, each site was cleansed of test material and five repetitive Sebutape samples collected at from lung fibrosis or internal malignancies. Thus, our data indicate that the presence of AHA is 30 min, 24 and 48 h post treatment. Sebutape was applied for 1 min, collected into vials and then classified as one of the serologic abnormalities observed in PM/DM. immediately frozen on dry ice. Tapes were thawed, extracted in 1 ml of saline and extracts assayed for cytokine levels using commercial immunoassay kits. Cytokine recoveries were normalized to total recovered protein. Results confirm the noninvasiveness of the Sebutape procedure as shown by lack of recovery of the inducible cytokine, IL-8 in any of 30 samples from a single site. The levels of normalized IL-101α were consistent across all 30 samples from two individuals ranging between 5 and 15 pg of IL-101α per ug total protein. The 24 h cytokine levels (IL-101α and IL-8) in SLS treated sites were significantly higher than naı¨ve sites with directional increases in normalized IL-101α and IL-8 levels, even in the absence of visible erythema. These data support continued development of this method as a noninvasive means to detect chemically induced inflammatory changes at the molecular level.

471 472 Use of Human Peripheral Blood Derived Dendritic Cells for Antigen Presentation in an In Vitro Role of Cytokines in Death Defying Phenotype of Psoriatic Keratinocytes (KCs) Lymphocyte Proliferation Assay J. Qin, V. Chaturvedi, P. Bacon and B. Nickoloff C. Ryan, B. Hulette and G. Gerberick Department of Pathology, Cardinal Bernardin Cancer Center, Loyola University, Chicago, Illinois The Procter & Gamble Company, Cincinnati, Ohio KCs in psoriatic plaques (PP) are exposed to cytokines and relatively resistant to apoptosis Dendritic cells (DC) are professional antigen presenting cells which are widely distributed in the compared to symptomless skin. To explore molecular events responsible for the antiapoptotic body and are key for initiating T cell mediated immune responses. Methods for culturing human phenotype of psoriaticKCs, proliferating human KCs were exposed for 8, 24, or 48 h to DCs from peripheral blood precursors have been developed by several laboratories. We were either:KGM alone, IFN-01γ, TNF-01α, TGF-01β, phorbol ester (TPA), or IFN-01γ plus TPA. interested in examining DCs produced by these methods and evaluating their ability to present Induction of apoptosis included exposure to UVB (30 mJ; 18 h) or trypsinization and suspension low molecular weight chemical haptens to sensitized T cells in an in vitro lymphocyte proliferation (48 h) in methylcellulose (MC). Apoptosis was assessed by flow cytometry with PI staining defined assay. Using peripheral blood from a dinitrochlorobenzene (DNCB) sensitized individual, immature by cells with sub-Go DNA content, and positive TUNEL staining. Only 1%–2% of proliferating DCs (iDCs), were generated by culturing T- and B-cell depleted mononuclear cells for 7 d in the untreated KCs (n ϭ 4) undergo spontaneous apoptosis, but after UVB exposure or suspension in presence of Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) and Interleukin-4 MC, 40%–60% of KCs were apoptotic While KCs exposed for only 8 h. to the various treatments (IL-4). Examination by flow cytometry demonstrated that these cells were MHC class II HLA- were not protected from apoptosis, KCs pretreated for 24 or 48 h did acquire a resistance to DRϩ, CD80ϩ, CD11bϩ, CD45ROϩ and CD36–. iDCs were treated with the either test allergen, induction of apoptosis, with the following representative percentages of apoptotic cell for each dinitrobenzenesulfonic acid (DNBS), a water soluble analog of DNCB, or an unrelated contact treatment (48 h) induced by UVB radiation; untreated (55%); IFN-01γ (12%); TNF-01α (23%); allergen, squaric acid (SA), then cultured with 105 autologous T cells at several responder (T-cells) TGF-01β (45%); TPA (4%); IFN-01γ ϩ TPA (2%). Since caspase cascades are important in to stimulator (DCs) ratios (R:S). Cultures were incubated for 4 d, pulsed with 3H-thymidine and apoptosis, levels of activated caspase 8, 3, and PARP cleavage were measured. After UVB exposure harvested 18–20 h later. T-cells cultured with DNBS haptenized iDCs at a 10:1 R:S demonstrated proliferating KCs underwent sequential changes at 4 h, 8 h, 16 h, 24 h – first displaying activation the greatest proliferation based on 3H-thymidine incorporation compared with control cultures. of caspase 8, then caspase 3, followed by PARP cleavage. However, in KCs pretreated with Mature DCs (mDCs) were generated from iDCs by culturing them in the presence of GM-CSF, cytokines, this caspase cascade was interrupted proportionate to the degree of protection from α β IL-4, TNF-01 , IL-101 , IL-6 and PGE2 for two additional days. When examined by flow apoptosis as measured by the PI staining/cell cycle analysis over the time course indicated. Indeed cytometry these cells, in addition to being HLA-DRϩ, were found to express high levels of CD80 for the best protective pretreatment combination of IFN-01γ plus TPA, the extent of blocking and CD86 and were also CD83ϩ. mDCs haptenized with DNBS were capable of stimulating a caspase activation was comparable to results obtained using potent synthetic caspase inhibitors (Z- proliferative response in sensitized T cells at an R:S of 50:1. Increased T cell proliferation above VAD, IETD). We conclude that certain cytokines can induce a striking antiapoptotic cytokine controls was also observed at an R:S ratio of 100:1. For both iDC and mDC experiments, no induced phenotype which requires approximately 24 h to develop in vitro, and involves blockage proliferation greater than control cultures of T-cells with untreated DCs was observed in T-cells of several key death effector molecules. Such a cytokine induced death defying phenotype may cultured with SA treated DC, the unrelated allergen, thus demonstrating the antigen specificity contribute to the antiapoptotic phenotype of PP skin and thereby the overall thickness of skin of the proliferative response to DNBS treated DC. These initial results show that mDCs are more induced by infiltrating pathogenic immunocytes that produce these cytokines. efficient antigen presenting cells for the chemical hapten DNBS.

473 474 Can Leukoeytes from Patients with Different Stages of Borreliosis and Morphea be Stimulated to Immune Complexes from Vasculitis Patients Bind to Endothelial Fc Receptors Independent of Produce Sulfidoleukotrienes in Response to Different Borrelia Genospecies? the Allelic Polymorphism of Fc01γRIIa U. Jappe and HL. Borkhardt, H Gollnick M. Gro¨ger, G. F. Fischer,* K. Wolff and P. Petzelbauer Department of Dermatology, Institute of Medical Microbiology, Otto-von-Guericke University Department of Dermatology, Division of General Dermatology and *Department of Blood Group of Magdeburg, Germany Serology, University of Vienna Medical School, Vienna, Austria The immunomechanisms controlling infections caused by Borrelia burgdorferi are only partially Cutaneous leukocytoclastic vasculitis is characterized by the deposition of circulating immune understood. Increased GM-CSF secretion and proliferation of PBMC in vitro have been shown complexes (CIC), neutrophil extravasation and vessel destruction, but mechanisms of CIC capture to be helpful in sero-negative cases. We therefore investigated 35 patients with confirmed within postcapillary venules are unknown. We demonstrate that CIC from sera of vasculitis borreliosis, 15 patients with morphea, a Borrelia-associated disease, and 24 healthy sero-negative patients bind to cultured endothelium in a Fc01γRIIa-dependent fashion. In lesional skin, controls for sulfidoleukotriene (SLT) production. Nineteen of 35 patients revealed Erythema endothelial cells bind IgG2ϾIgG3 and IgG4, but not IgG1, even before obvious neutrophil chromicum. migrans (ECM), and 16 of 35 later stages of infection. Sedimented leukocytes from transmigration and vessel damage. Since the human Fc01γRIIa proteins exist in two allotypes, patients’ blood were stimulated with ultrasonicated sterile-filtrated Borrelia burgdorferi (B) sensu one with a histidine at position 131 (131H), which binds IgG1, 2, 3 and the other with an stricto, B. garinii, and B. afzelii antigens. An ELISA based on a monoclonal antibody that recognizes arginine at position 131 (131R), which binds IgG1, and 3, but is unable to bind IgG2, we different types of leukotrienes equally well was performed. B. afzelii stimulated much better than expected a prevalence of 131H forms in vasculitis patients. However, sequence analysis revealed the other two genospecies. Patients with later stages of borreliosis revealed elevated SLT levels in an equal distribution of allotypes in patients and controls. In conclusion, CIC binding to endothelial contrast to ECM and controls. Comparison of patients before and after therapy showed significantly Fc01γRIIa is among the initial steps in the development of vasculitis. Although IgG2 is the higher production post rather than pretherapy when stimulated with B. afzelii. For the first time predominant subtype precipitated at endothelial surfaces, it is not required for fixing CIC to we could show that there is an in vitro SLT production of leukocytes from Borrelia infected patients, endothelium, because patients homozygote for Fc01γRIIa-R131 equally develop LV as those in contrast to morphea and controls, which is species-, stage- and therapy dependent. This may bearing the Fc01γRIIa-H131 allele. Since IgG1 is virtually absent in LV lesions and IgG4 does be explained by the increase of an in vivo stimulation of leukocytes due to persistent Borrelia infection not bind to both Fc01γRIIa alleles, these complexes, in addition to IgG2, must contain IgG3 in order to fix to vascular Fc01γRIIa, at least in persons homozygote for Fc01γRIIa-R131. 602 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

475 476 Failure of Foreign and Self Antigen Peptides of the NC16A Domain in the Induction of The Role of Eotaxin, an Eosinophil-Specific Chemokine, in the Blistering Process of Incontin- Autoimmunity to BPAg2 in BALB/C Mice entia Pigmenti L. Xu, N. Robinson, S. D. Miller and L. S. Chan S. Jean-Baptiste, J. Guitart, A. Paller and L. S. Chan Departments of Dermatology and Microbiology and Immunology, North-western University of Department of Dermatology, North-western University Medical School, Chicago, Illinois Medicine School, Chicago, Illinois Incontinentia pigmenti (IP) is an X-linked dominantly inherited disease. In the first stage, vesicular We attempted to induce an active model of murine bullous pemphigoid (BP) by subcutaneous lesions are observed clinically and intraepidermal blister with prominent eosinophil infiltration are immunization with antigenic peptides of human and murine BPAg2 NC16A domain, in order to observed histopathologically. The pathomechanism accounting for the recruitment of eosinophils understand autoimmune induction in BP. Two overlapping peptides were synthesized in the form into the epidermis is unknown. Eotaxin, a newly characterized, epithelial cell-produced, eosinophil- of a multiple peptide antigen on a solid lysine base. Female BALB/C mice were injected with specific chemokine, may play a role. We examined the epidermal expression of eotaxin in the IP three varying doses of peptides (human, murine, or combined human and murine), or phosphate- epidermis. Immunohistochemistry was performed on skin biopsies of patients with the vesiculobull- buffered saline control emulsified in complete Freund’s adjuvant, on a four-week interval. At the ous stage of IP (N ϭ 13), noneosinophilic skin inflammation (eczema, N ϭ 7; dermatitis third immunization, half of the mice in each group were given three doses of 10,000 units of herpetiformis, N ϭ 3), and normal individuals (N ϭ 8), using a monoclonal antibody against IFN-alpha, in addition to the peptides, in order to enhance antigen presentation. For the fourth human eotaxin. In additon, Giemsa stains were performed to identify the cellular infiltrates. Our immunization, all the mice that previously received human peptides were given murine peptides study results revealed that normal epidermis constitutively expressed a low level of eotaxin, similar in order to test whether priming with a foreign antigen (human BPAg2) would break the tolerance to the level of expression in the epidermis of noneosinophilic skin inflammation. In contrast, the to self antigen (murine BPAg2). Five weeks after the fourth immunization, all mice failed to show vesiculobullous stage of IP skin showed a strong expression of eotaxin throughout all layers of the skin blisters, as well as in vivo-bound or circulating IgG autoantibodies to the mouse skin basement epidermis, particularly in areas adjacent to the blisters. Furthermore, Giemsa stains confirmed the membrane. Failure of the induction of BP could be due to the following: (i) failure of the peptide prominent epidermal eosinophil infiltration and demonstrated a large number of dermal mast cells antigens to induce autoimmunity to BPAg2; (ii) genetic resistance of BALB/C mice to autoimmune in most IP specimens. These data suggest that epidermal eotaxin may play a role in recruiting induction; (iii) antigenic competition between peptides within the overlapping set. eosinophils into the IP epidermis. Cytokines produced by mast cells, such as IL-1 and TNF-alpha, may play a role in activating epidermal cells to synthesize and secrete eotaxin.

477 478 Depressed Interleukin-12 Mediated Signal Transduction in T-Cells from Patients with Sezary Growth Hormone Enhances Epidermal Growth Factor Receptor Expression on Keratinocyte in β Syndrome is Associated with the Absence of IL-12 Receptor 01 2 mRNA Expression a CO2 Concentration Dependent Manner L. Showe, F. E. Fox, Z. Niu, D. Williams and A. H. Rook T. Takahashi, I. Ando, A. Kukita and K. Tamaki Department of Dermatology, University of Pennsylvania and the Wistar Institute, Philadelphia, Department of Dermatology, The Mizonokuchi Hospital of Teikyo University, Kanagawa, Japan, Pennsylvania and Department of Dermatology, The University of Tokyo, Tokyo, Japan Sezary syndrome (SS) is the leukemic phase of Cutaneous T-cell Lymphoma (CTCL) characterized Acquired pachydermoperiostosis is sometimes caused by growth hormone (GH) secreting adenocar- by the proliferation of clonally derived CD401ϩ T-cells which release cytokines of the Th2 T- cinoma. This disease exhibited acanthosis in distal site of fingers as well as proliferation of collagen cell phenotype (IL-4, 5, 10). Production of Th1 cytokines (IL-2, IFN-01γ) is markedly depressed fiber bundles and periostal new bone formation. Aanthosis is also observed in acromegaly patients. as is expression of IL-12, a pivotal cytokine for Th1 cell differentiation. Since the 01β2 chain of The findings suggest direct or indirect proliferation effect of GH on keratinocytes. This has not the IL-12 receptor (IL-12R) is required for IL-12 signal transduction, and is absent on Th2 cells, clearly been elucidated. We initially certified that GH did not directly enhance the proliferation we examined message levels of the IL-12R 01β1 and 01β2 genes in patient lymphocytes by of keratinocytes in vitro, as was previously reported. Next, we studied the effect of GH on receptors RNAse protection assays in seven SS patients, with Ͼ85% of their CD4ϩ T-cells being Sezary expression of cytokines and growth factors related with proliferation of keratinocytes. By FACS cells. 01β2 mRNA was low to undetectable in six patients and high normal levels in one patient. analysis, GH enhanced receptor expression of epidermal growth factor (EGF) but not those of IFN-01α and IFN-01γ, known inducers of 01β1 and 01β2, stimulated expression of both genes insuline-like growth factor, fibroblast growth factor, IL-1, IL-6, IL-8. Such enhancement was α β β in normal lymphocytes, but IFN-01 had no effect on either 01 1or01 2 expression in SS cells more prominent in keratinocytes incubated under low concentrations of CO2 (0%–2%) condition. and only 01β1 was induced by IFN-01γ. Normal Th2 cells that don’t express 01β2, still bind IL- By [3H]-thymidine uptake assay and cell cycle analysis, keratinocytes incubated with GH and 12 but fail to phosphorylate Stat4. SS cells not only fail to phosphorylate Stat4 in response to IL- EGF actually showed the higher proliferation activity compared with the keratinocytes only with 12, but levels of Stat4 protein are severely reduced in these cells as compared to normal controls. EGF. On the other hands, no significant enhancement was observed in proliferation of keratiocytes This is the first reported observation of a defect in Stat4 gene expression associated with a human with GH and other cytokines or growth factors. These results suggest high GH level may fill a disease. Moreover, these data further suggest that T-cells in SS are of the Th2 phenotype. role in the formation of acanthosis in acquired pachydermoperiostosis through enhancement of Understanding the mechanism which results in suppression of Stat4 in SS cells may be significant EGF receptor expression. to understanding the defining events in the development of SS and related CTCL.

479 480 The RNA Splicing Regulator Transformer-2 (Tra-2) as an Antigenic Target of Autoantibodies Adenosine Deaminase as an Antigenic Target of Autoantibodies from Patients with Systemic from Patients with Systemic Rheumatic Diseases Lupus Erythematosus J. Deng, J. Hempel, R. Frye and W. Mattox J. Lee, J. Hempel, S. Manzi and J. Deng Department of Dermatology, University of Pittsburgh and VAMC, Pittsburgh, Pennsylvania, and University of Pittsburgh and VAMC, Pittsburgh, Pennsylvania Department of Molecular Biology and Genetics, the University of Texas, Maryland Anderson Adenosine deaminase (ADA) is an enzyme involved in purine metabolism and present in all Cancer Center, Houston, Texas mammalian tissues. It appears to have a major role in the development and function of lymphoid Transformer-2 (Tra-2) is a RNA binding protein that regulates sexual differentiation in Drosophila cells. Congenital deficiency of ADA is characterized by the absence of functional mature T and through effects on alternative premRNA splicing. Human homologues of Tra-2 are also thought B lymphocytes. Patients with congenital ADA deficiency treated with precipitated ADA result in to regulate alternative splicing. Several RNA splicing factors have been identified which are development of antibodies to ADA suggesting that ADA molecules might be immunogenic. This antigenic for autoantibodies detected in patients with systemic rheumatic diseases. Since Tra-2 is let us speculate the possible presence of anti-ADA antiboodies and its relationship to lymphopenia related to this group of antigenic factors, we initiated a study to evaluate the significance of in patients with systemic rheumatic diseases. Commercial available ADA was used in ELISA and antibodies to Tra-2 in patients with systemic rheumatic diseases. Recombinant human Tra-2beta immunoblots for detection of anti-ADA antibodies. Four out of 50 patients examined were was used as antigen in ELISA and immunoblots performed with sera from 50 patients to screen positive for anti-ADA antibodies. Two of them had peripheral blood lymphopenia, while the for the presence of anti-Tra-2 antibodies. The results were further compared with that obtained other two had normal lymphocyte count. ADA-nitrocellulose was used to purify anti-ADA from Hela cell extracts as the antigenic source. Sera from six patients were found to have reactivity antibodies. The purified antibodies were used to locate the distribution of ADA on Hep-2 to Tra-2beta in ELISA as well as in immunoblots. However, these sera had minimal reactivity to and lymphocytes. Anti-ADA antibodies gave distinct nuclear speckled pattern under indirect Tra-2 in Hela cell extracts, most likely because Tra-2 is expressed at low levels relative to other immunofluorescence. Anti-ADA also stained the cell surface of Hep-2 cells and lymphocytes on proteins such as snRNPs. Polyclonal anti-Tra-2 antibodies were purified and used to determine viable cell immunofluorescence. The anti-ADA antibodies fail to gain access into cytoplasm or the distribution of Tra-2 in Hela cells. Surprisingly, Tra-2 was found in the nucleoli of these cells. nuclei when added to with viable Hep-2 cells or lymphocytes. This study indicates that anti-ADA This study indicates that Tra-2beta is one of the antigenic molecules to which autoantibodies are antibodies may be present in patients with systemic rheumatic diseases and may not be associated produced in patients with systemic rheumatic diseases with lymphopenia. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 603

481 482 Expression of Antimicrobial Peptides in Human Oral Keratinocytes and Tissue Severe Atypical Psoriatic Erythroderma Precipitated by Acute Hepatitis B Infection B. A. Dale, S. Krisanaprakornkit and J. R. Kimball and A. Weinberg K. Koo, M. Lebwohl and R. Forney Department of Oral Biology and Medicine/Dermatology, University of Washington,. Seattle, Departments of Dermatology, UCSF Medical Center, San Francisco, California, and Mt. Sinai Washington and Department of Periodontics, Case Western Reserve University, Cleveland, Ohio Medical Center, New York, New York Skin and mucosal epithelia act as physical barriers to protect the host from the environment via A 21-y-old Asian male with psoriasis well controlled by methotrexate missed four doses of their tightly adherent architecture and specialized structural proteins. In addition, they express methotrexate. He presented with an expected flare of his usual plaque-type psoriasis and was 01β-defensin antimicrobial peptides, components of the innate host defense system, that act on a restarted on his full methotrexate dose. He was initiated on systemic PUVA therapy and received broad spectrum of microorganisms. We hypo-thesize that antimicrobial peptides are important in three treatments up to 3 J. Three days after the second dose of methotrexate, he presented with maintaining the balance between health and disease in the oral cavity which is constantly exposed explosive onset of erythroderma including extensive painful erosions of his psoriatic skin. He was to microbial challenges. The purpose of this study was to determine expression of human 01β- admitted to the hospital where he was found to have an elevated white blood count, liver defensin-1 and Ϫ2 (hBD-1,-2) in gingival epithelia. Keratinocytes from normal gingiva (GECs) functions tests, and glucose. Previously his only medical problem was psoriasis. Biopsies of the were challenged with cell wall extract from Porphyromonas gingivalis (Pg; pathogenic) or Fusobacterium erosions and newly erythrodermic skin showed ‘‘pustular’’ psoriasis, though clinically no pustules nucleatum (Fn; nonpathogenic), E. coli lipopoly saccharide, TNF01α, and phorbol myristate acetate were present, but not drug eruption or photosensitivity reaction. He was discharged after 5 d and (PMA) were analyzed by RT-PCR. HBD-2 mRNA was upregulated by TNF01α and Fn within began on cyclosporine and outpatient Goeckerman treatment. Due to the elevated liver function 2–4 h, and by PMA after 10 h, but poorly upregulated by other stimuli. In contrast, hBD-1 tests, a full hepatitis panel was run. Serum was positive for hepatitis B IgM as well as hepatitis B core mRNA was previously shown to be constitutively expressed in all conditions (Krisanaprakornkit antibody. Cyclosporine was immediately discontinued. He eventually cleared with Goeckerman and et al, Infect Imm 66:4222–8, 1998). However, individual variation in hBD-1 and hBD-2 mRNA bath PUVA therapy. levels was observed in gingival tissue samples (n ϭ 15). Monoclonal antibody to hBD-1 showed It is well known that acute infection frequently exacerbates psoriasis. This case is being presented greatest peptide expression at the gingival margin, the region of gingiva adjacent to the tooth as the first reported case of psoriasis exacerbation that may have been due to acute hepatitis B. which is exposed to plaque bacteria. In GECs, only samples stimulated with Fn showed detectable Given the increasing number of cases of hepatitis of all types, this case encourages us to think of immunoreaction. Immunoreactivity suggests post-transcriptional regulation of hBD-1 peptide acute hepatitis as a possible cause when a clinician is faced with a sudden, atypical, unexplained expression. Results indicate that hBD-1 and Ϫ2 are differentially upregulated in gingival flare of psoriasis. Determining the presence or absence of acute hepatitis is also critical in making keratinocytes in response to inflammatory stimuli, and implicate a role in health and susceptibility appropriate treatment choices. to disease.

483 484 Antimicrobial Activity of a Novel Starch-Oil Topical Delivery System, Fantesk Human Immunodeficiency Virus Infected Cells Increase in Skin After Low Dose Ultraviolet B F. O. Cope and J. J. Wille Treatment and Negatively Correlate with Peripheral Load Hy-Gene, Inc. and 36 Broad Street, Trenton, New Jersey J. Breuer-McHam, D. E. Lewis and M. Duvic Fantesk is a novel patented nonseparable starch-oil composite that is formed by a jet-cooking Departments of Dermatology and Internal Medicine, U. Texas Medical School; Section of process. The emulsion is a two-component system in which solubilized starch is the bulk phase Dermatology, MD Anderson Cancer Center; and Department of Microbiology & Immunology, and the oil phase forms a stable dispersion within the starch matrix. No emulsifiers or stabilizers Baylor College of Medicine, Houston, Texas are required to maintain this stable emulsion. We report the results of our studies aimed to explore To determine the effect of UVL on HIV levels we studied skin and serum in patients with AIDS- the use of this starch-based gel material as a topical delivery system for antimicrobial fatty acids. related skin diseases, psoriasis and pruritus, and irradiated an HIV transfected Jurkat T cell line A number of different gel formulations were produced including a base formulation composed of with doses of UVB. Skin and peripheral viral load were measured in 10 HIVϩ patients over 6 from 10% to 20% starch solids and between 0.5% and 5% lipids in water. The starch-oil composite wk of phototherapy, using RT-PCR in situ hybridization and RT-PCR, respectively. Numbers vehicle was a base formulation containing 10% (wt/vol) starch and 5% (wt/vol) soybean oil in of peripheral HIV-RNAϩ skin cells were compared to levels of HIV-RNA in serum. The median water. The active formulation was a starch-oil composite that contained 10% (wt/vol) starch and number of gag positive cells per mm2 in skin lesions doubled from 1.1 at baseline to 2.3 at 2 wk 1% palmitoletic acid in water. In order to examine the specificity of the fatty acid antimicrobial, of UVL (Ͻ1000 mJ per cm2) and was negatively correlated with decreased serum HIV-RNA a starch-oil composite containing 1% oleic acid was also tested. The following antimicrobial test (Pearson correlation ϭ –0.799, P ϭ 0.017). However, after 6 wk of phototherapy, the number assay was developed and used for these studies: Whatman no. 4 circular filter paper discs (4 inch of infected cells in skin lesions (median ϭ 0.3 cells per mm2) decreased relative to baseline number diameter) were cut into small wedges 2 cm wide at the semicircular circumference tapering to (median ϭ 1.1 cells per mm2) of infected cells, and skin lesions improved. Serum HIV-RNA zero width at the apex. The cut paper was sterilized by autoclaving and individual pieces placed increased in five of 10 patients relative to baseline (median ϭ 33,000, change from 1300 to in a dry Petri dish. Each piece was wetted with sterile Soybean Trypticase broth (STB). The 23,000). In an HIV ϩ Jurkat cell model, HIV transcription peaked at 50–100 J per m2 of UVB wetted papers were then loaded with either a 50 µl aliquot containing either IXPBS (negative at 24 h. and was reduced on either side of this dose. This indicates that the dose of UVB is control) or a 1:100 dilution of IXPBS(pH 7.2) containing 2 01ϫ 10 8 Bacillus subtilis bacteria important in influencing HIV expression in skin, periphery, and in an in vitro model system. which had previously been cultivated overnight in STB broth to yield exponential cultures. An aliquot of this culture was removed, and thoroughly washed in IXPBS by low speed centrifugation and serial dilution in 1XPBS. The bacteria were allowed to soak into the wetted papers, and the various test gels applied for 30 min, after which they were dropped aseptically into fresh sterile SBT broth and the cultures shaken overnight at room temperature. The cell density of bacterial of control and experimental cultures were put into 96-well plates and read in a automated plate reader. In many independent trials, no growth occurred in culture flasks inoculated with bacterial papers treated with starch-oil gels containing palmitoleic acid. Aside from the negative (no bacteria) control, all other treatment groups blossomed into bacterized cultures

485 486 Infection of Dendritic Cells (DCs) with Human Herpesvirus-8 (HHV-8) Famciclovir Versus Acyclovir for the Treatment of Ophthalmic Herpes Zoster K. Foreman, J. Kash, D. Eilers and B. Nickoloff T. Evans, S. Tyring, R. Engst, M. Lassonde, S. Trottier, S. Van Slycken, R. Crann, L. Locke and Departments of Pathology, Medicine, and Dermatology and Cardinal Bernardin Cancer Center, A. Palestine Loyola University, Maywood, Illinois UTMB, Galveston, Texas; Dermatological Hospital, Munich, Germany; Hospital of Notre Dame, Recent studies indicate the DNA virus implicated in Kaposi’s sarcoma (KS) called HHV-8 can Montreal, Quebec, Canada; Centre Hosp. De L’Universite Laval, Ste.-Foy, Quebec, Canada; infect a variety of cell types in vivo including spindle shaped KS tumor cells, DCs, endothelial cells Gent, Belgium; SmithKline Beecham, Collegeville, Pennsylvania; Georgetown University, Wash- (ECs), and B-cells. HHV-8 has also been localized to DCs in multiple myeloma (MM). Since KS ington, DC and MM represent highly distinctive and vastly different neoplasms, we proposed a unifying Ophthalmic herpes zoster (HZO) is one of the most serious manifestations of varicella-zoster virus hypothesis that involved HHV8 infection of DCs that could drive local proliferation in a paracrine infection. Up to 72% of patients have complications involving ocular structures, which can lead fashion of ECs in the skin to produce KS and B-cells/plasma cells in bone marrow to produce to ocular events ranging from self-limited processes to chronic ocular inflammation or neuropathy MM. As an initial attempt to validate this hypothesis, we attempted to infect DCs from peripheral and subsequent visual loss. Anterior uveitis and keratitis are common (up to 56% of patients; blood with HHV-8. Peripheral blood mononuclear cells were isolated, adhered to plastic, and Cobo, et al, 1986) long-term complications contributing to vision loss in untreated patients. In cultured in GM-CSF/IL-4 to differentiate cells along a DC pathway. Two weeks later, DCs were view of these serious complications, appropriate, timely treatment of HZO is advocated. Acyclovir infected with purified HHV-8 (i.e. cell-free) derived from the BC-3 cell line, and cultured for an (ACV) has been shown to be beneficial in the treatment of HZO and is currently the standard of additional 7 d. Infected as well as uninfected control cells were photographed and processed for care. However, poor and variable ACV bioavailability requires frequent dosing, which can lead RNA isolation and/or electron microscopy (EM). Morphologic examination of HHV-8 infected to noncompliance. The current study, the largest randomized, double-blind, controlled HZO trial DCs demonstrated the conspicuous presence of multinucleated giant cells as well as cells containing conducted to date, compares famciclovir (FCV) 500 mg tid with ACV 800 mg five times daily lipid droplets and vacuoles which were absent in uninfected control cultures. RT-PCR confirmed for 7 d in the treatment of HZO. The groups (251 FCV; 203 ACV) were well matched for the DCs were infected with HHV-8 as transcripts for ORF73 (LANA) and ORF22 (glycoprotein baseline characteristics. The population was 53% female and 95% white; mean age was 58 years. H) were detected in the infected, but not uninfected control cells. Transcripts for ORF16, which All patients enrolled within 72 h of rash onset. Results for the primary endpoint of proportion of are expressed only during lytic infection, were not detected. Intact viral particles were not readily patients with ocular manifestations during the study demonstrated that FCV and ACV were of apparent on EM suggesting that the majority of cells are latently infected. In conclusion, these similar clinical efficacy (58.0% FCV; 58.2% ACV). Severe ocular manifestations occurred in a results indicate that DCs can be infected by HHV-8 in which HHV-8 is maintained in a latent similar proportion of patients in each group. Anterior uveitis occurred in only 25% of FCV and state. This result is consistent with previous studies indicating that HHV-8 is present in low copy ACV recipients; keratitis occurred in only 13.2% of FCV and 18.4% of ACV recipients. Few number in cells not undergoing lytic replication, but rather undergoing latent infection, which patients experienced a loss in visual acuity (2.6% FCV; 6.3% ACV). The adverse event and clinical has been observed in vivo at tissue sites undergoing cellular transformation by HHV-8. laboratory profiles of the two treatments were similar. In summary, FCV 500 mg tid for 7 d is as effective and well-tolerated as the current standard of care in HZO treatment but with more convenient dosing. 604 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

487 488 Cidofovir Regulates Expression of Various Cyclins in Human Papillomavirus-Infected Keratinocytes Characterization and Rapid Quantification of Human Herpesvirus 8 Lytic Replication in I. Arany, M. Brysk, C. Arrastia and S. Tyring Single Cells: Implications for Designing Treatment for and Understanding Pathogenesis of Department of Microbiology/Immunology and Dermatology and Obstetrics/Gynecology, The Kaposi’s Sarcoma University of Texas Medical Branch, Galveston, Texas J. Zoeteweij, S. Eyes, T. Kawamura, B. Chandran, B. Forghani, S. Cohen and A. Blauvelt Cidofovir (HPMPC) demonstrated remarkable antiviral effects on a variety of DNA viruses. Dermatology Branch, NCI and Howard Hughes Medical Institute, Bethesda, Maryland; University HPMPC is believed to inhibit viral DNA polymerase. Clinical studies showed that HPMPC is of Kansas, Kansas City, Kansas; California Department of Health Services, Berkeley, California also potent in treatment of genital warts induced by human papillomaviruses (HPVs). Clinically, Human herpesvirus 8 (HHV8) infection is associated with Kaposi’s sarcoma (KS), primary effusion Cidofovir exerted activity against anogenital warts with low recurrence rate in HIV-infected lymphoma (PEL), and certain forms of Castleman’s disease. Detection and quantification of single individuals. Certainly, the mechanisms of action on HPV are not identical to those against herpes cells infected with HHV8 is currently limited and technically difficult. In this study, we used viruses since HPV does not encode its own polymerase. Rather, HPMPC might interact with newly developed mAbs and flow cytometry to detect HHV8 lytic-phase antigens encoded by various cellular genes that are important in viral replication and transcription. Candidates are the ORF59 (early lytic PF-8 protein) and ORFK8.1 (late-lytic envelope gp35/37 protein) in phorbol members of the cyclin system and its related kinases that regulate cell cycle progression and are ester (TPA) or butyrate-stimulated HHV8ϩ PEL cell lines (BCBL-1 or BC-3). Approximately necessary for HPV activities. Earlier we found that progression of low-grade cervical dysplasias 0.2% and 0.8% of unstimulated PEL cells were positive for gp35/37 and PF-8, respectively. After (LCIN) to high grade ones (HCIN) was accompanied by increased expression of cyclins and 2 d induction with TPA (0.03 mM) or butyrate (0.3 mM), 0.9%–6.5% of viable PEL cells cyclin-dependent kinases. Accordingly, we treated an HPV 16-transformed cervical carcinoma expressed gp35/37, while 9.4%–21% expressed PF-8. We then used our assay to screen potential line (SiHa) with Cidofovir (20 mg per ml) for various times (2–24 h) in vitro. The treatment antiherpesviral agents, by adding drugs to stimulated PEL cultures. Acyclovir, foscarnet, cidofovir, resulted in a marked decrease in cyclin B (75%) and cyclin E (60%) levels as revealed by a and PMEA all reduced the number of cells expressing gp35/37 in a dose-dependent manner, but semiquantitative RT-PCR method. Levels of cdc2 and cdk2 kinases were also diminished (40% not the number of cells expressing PF-8, showing that these drugs inhibited viral structural protein and 45%, respectively). These kinases phosphorylate cyclins at various points of the cell cycle. synthesis as expected, but not early lytic phase antigen expression. By contrast, the antiviral Cyclin B and E are important in HPV-mediated cell cycle disruption. Thus, downregulation of cytokines IFN01α, IFN01β, and IFN01γ reduced the number of cells positive for both PF-8 and the activities of those genes has great therapeutic potential and might be involved in the observed gp35/37, consistent with an overall inhibitory effect on viral gene transcription/translation. In clinical activity of Cidofovir against anogenital warts. summary, we have developed a novel technique that allows rapid quantification of HHV8 lytic infection in single cells. This assay should prove extremely useful in screening and characterization of possible antiherpesviral agents, as well as in the detailed phenotypic characterization of HHV8- infected cells in patients with KS.

489 490 Epstein-Barr Virus Infection in Patients with Vitiligo The Identification and Determination of Active Growth of Dermatophytes Based on the Actin Gene P. E. Grimes, T. Elkadi and J. Sanders C. Okeke, R. Tsuboi, M. Kawai and H. Ogawa Savall. Vitiligo and Pigmentation Institute of Southern California Los Angeles, California Department of Dermatology, Juntendo University School of Medicine, Tokyo, Japan A previous study has suggested that a viral agent may be involved in the pathogenesis of vitiligo. Polymerase chain reaction (PCR) and reverse transcription-3’rapid amplification of cDNA ends- Epstein-Barr virus (EBV) has been implicated in the pathogenesis of a variety of autoimmune nested PCR (RT-3’RACE-nested PCR) amplification of the actin gene (ACT) were used to diseases. This investigation was undertaken to assess Epstein-Barr virus seroconversion and antibody identify and evaluate the growth activity of dermatophytes in cultures and skin scrapings. A 750- titers from sera of patients with vitiligo. Eighty nine patients with vitiligo were included in this bp DNA fragment was nonspecifically amplified by PCR from the genomic DNA extract of study. Their mean age was 36 Ϯ 2.0 SE. The mean duration of the disease was 8 Ϯ 1.2 SE. Trichophyton mentagrophytes employing primers based on the nucleotide sequence of Candida albicans Forty-nine age, race and sex matched control subjects were also included in this investigation. An ACT. Database search indicated that the nucleotide and deduced amino acid sequences from the immunofluorescence assay (Specialty Laboratories, Los Angeles, California) was used to detect the fragment contain regions homologous to the conserved sequences of actin gene. Fragments were presence of EBV antibodies. Patients and controls were screened for EBV early antigen, IgG and amplified by PCR from 16 different species of dermatophytes and sequenced. Specific detection IgM viral capsid antigens, and EBV nuclear antigen. There were no statistically significant of ACT mRNA by RT-3’RACE-nested PCR in total RNA extracts from cultures and samples differences in seroconversion or antibody titers for EBV-VCA IgG, IgM or EBV nuclear antigen. from skin scrapings was employed to detected dermatophyte growth activity. The dermatophyte VCA-IgG seroconversion was 88% and 85% for patients and controls, respectively. In contrast, species were distinguished by PCR using ACT primers based on species-specific nucleotide 45% of the patients had detectable EBV early antigen compared to 16% of control subjects (P Ͻ sequences. Detection of ACT mRNA correlated with fungal growth activity or viability. In 0.01). Mean EBV early antigen titers were 105 Ϯ 19 SE as compared to 19 Ϯ 4 SE in the control conclusion, PCR and RT-3’RACE-nested PCR amplification of ACT enables identification and subjects (P Ͻ 0.05). This data suggests that EBV infection in patients with vitiligo may be assessment of growth activity of dermatophytes. associated with a chronic active infection characterized by persistent ongoing viremia. In light of immunodysregulation vitiligo patients may have a unique susceptibility to chronic EBV infection. Alternatively, EBV infection may predispose genetically susceptible patients to vitiligo and autoimmunity.

491 492 In Vitro Absorption and Penetration of UV Filters on Fresh Excised Pig Skin Electron Paramagnetic Resonance Study Utilizing Stripping Method on Normal Human Stratum M. T. Fernańdez, H. -J. Duesing, J. L. P. Juez* and W. Diembeck Corneum In Vivo Biocompatibility/Biochemistry Department, Research cosmed, Beiersdorf AG, Hamburg, Germany J. Mizushima, Y. Kawasaki, T. Kitano, K. Sakamoto, M. Kawashima, R. Cooke and H. I. Maibach and *Research and Development Center, CSIC, Barcelona, Spain Department of Dermatology, University of California, San Francisco, California; Amino Science Since dermal absorption and percutaneous penetration of cosmetic ingredients depend on a Laboratories, Ajinomoto Co., Kawasaki, Japan; Department of Dermatology, Tokyo Women’s multitude of variable factors, like vehicle composition, application area dose, exposure time, Medical University, Tokyo, Japan; Department of Biophysics and Biochemistry, University of species and skin preparation, skin temperature and others, only data obtained in accordance with California, San Francisco, California the same test protocol should be compared. Electron Paramagnetic Resonance (EPR) with nitroxide spin probe was used to assess the fluidity Following this protocol, we have carried out absorption/penetration studies on Caffeine and of normal stratum corneum (SC) of in vivo human skin, removed from the forearm by a single five sunfilters (octyl methoxycinnamate, benzophenone-3, benzophenone-4, octyl triazone and stripping with cyanoacrylate resin, onto quartz glass, cover glass or transparent film. Spectra were octocrylene) in an oil/water emulsion using split-thickness pig skin. compared with those of SC of cadaver skin. All stripped SC spectra were similar to those of Fresh skin from six different animals was used in triplicate to study the intra- and interindividual cadaver skin. There was no statistical difference in order parameter S between SC of cadaver skin variability of the repartition profiles of each test substance. After 16 h exposure time in Franz and those on quartz cell and cover glass. Those on transparent film were significantly higher. static diffusion cells, the excessive emulsion remaining on the skin surface was recovered by Spectra after a months storage at room temperature, at 50°C, and at –20°C, were unchanged; S washing. Thereafter, 16 strips were taken with D-Squame by applying a constant pressure during was decreased after a 3-mo storage in all conditions. The results suggest that the in vivo stripping a defined time and epidermis was separated from dermis by heat treatment. Amounts of test method is useful for the evaluation of skin conditions by means of fluidity measurement of in vivo substances in the stratum corneum (strips), epidermis, dermis, receptor fluid and washing solutions SC utilizing EPR were determined by HPLC analysis (diode array detector). UV filters yielded different distribution profiles in the stratum corneum as well as in epidermis and dermis and none of them was detected in the receptor fluid. The intra-and interindividual variability was found to be 12% and 17%, respectively. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 605

493 494 Effect of Permeability Barrier Disruption on the Percutaneous Penetration of Drugs With Occlusive Therapy in Psoriasis; Pathogenetic Roles of Permeability Barrier and Calcium Various Lipophilicity S. Hwang, S. Jiang, G. Menon,* E. Choi, S. Ahn and S. Lee† J-C. Tsai, P-L. Hung and H-M. Sheu Department of Dermatology, Yonsei University Wonju College of Medicine, Wonju, Korea; Institute of Clinical Pharmacy and Department of Dermatology, National Cheng Kung University *Avon Products, Inc., Suffern, New York; and †Yonsei University College of Medicine, Medical College, Tainan, Taiwan Seoul, Korea The objective of the study was to investigate the effect of permeability barrier disruption by Although occlusive dressings have great potential in the management of psoriasis vulgaris, the acetone extraction of the stratum corneum lipids on the percutaneous penetration of drugs with therapeutic mechanism is not completely understood. To investigate the pathogenetic roles of the various lipophilicity. Hairless mice were treated topically with absolute acetone by gently and permeability barrier and calcium on the therapeutic effects of occlusion, we studied the permeability repetitively rolling solvent-soaked cotton balls on one flank until transepidermal water loss (TEWL) barrier function, intercorneocyte lipid layers, lamellar bodies, and calcium gradients in chronic reached designated levels (up to 40 g per m2 per h), as measured with Tewameter. The area were plaque type psoriasis after occlusion with water vapor-impermeable membrane. The transepidermal then demarcated and the full thickness skin excised for flux measurement of caffeine (log K ϭ – water loss (TEWL) was measured and skin biopsies were taken at preocclusion, 3 and 7 d with 0.02), hydrocortisone (log K ϭ 1.5), estradiol (log K ϭ 2.7), and progesterone (log K ϭ 3.9) occlusion. The specimens were processed by ruthenium tetroxide postfixation and ion-capture using flow-through diffusion cells. The results demonstrated that, when the TEWL of hairless cytochemistry for electron microscopy. With improvement of clinical features, TEWL was mouse was elevated from 2.8 to 6.8 g per m2 per h (normal untreated) to 40 g per m2 per h after significantly decreased at 3 d with occlusion and maintained for 7 d without returning to the acetone treatment, the skin permeability of caffeine increased linearly with the degree of barrier normal range. Occlusion for 3 d produced a decrease of unfurled and whorled pattern of disruption. Among the four drugs, percutaneous penetration enhancement for hydrocortisone due intercellular lamellae structure in the stratum corneum interstices. Occlusion for 7 d revealed a to barrier disruption was the most dramatic that its skin permeability increased log-linearly with nearly mature pattern of intercellular lamellar structures and produced partial re-establishment of TEWL elevation. For estradiol, there appeared to be a linear correlation between TEWL and it’s epidermal calcium gradients and disappearance of calcium precipitates from the stratum corneum skin permeability when TEWL was less 10 g per m2 per h. The increase in TEWL beyond that interstices. These results provide that the normalization of the permeability barrier and epidermal level didn’t result in further increase in skin permeability of estradiol. The skin permeability of calcium gradients play important roles in the therapeutic effects of occlusive dressing in progesterone remained relatively unchanged through the entire range of barrier disruption. We psoriasis vulgaris. have concluded that permeability barrier disruption by acetone treatment enhanced significantly the skin permeability of both hydrophilic and amphipathic drugs. The treatment didn’t appear to alter the percutaneous penetration of highly lipophilic drugs.

495 496 Peroxisome Proliferator-Activated Receptor-01α (PPAR-01α) Enhances Lipid Metabolism in a Anatomically Identifying the Shunt Pathway of Human Sweat Glands During Electrofacilitated Skin Equivalent Model Transcutaneous Drug Delivery M. Rivier, I. Castiel,* I. Safonova, G. Ailhaud† and S. Michel W. Chen GALDERMA R&D, Sophia-Antipolis; *L’OREAL, Centre C. Zviak, Clichy; and †Laboratoire Department of Dermatology, the University of Illinois, School of Medicine, Chicago, Illinois de Biologie du Tissu Adipeux, Nice, France The importance of skin appendages as low-resistance shunt pathways during electrofacilitated Peroxisome proliferator-activated receptors (PPARs) are involved in the control of several pathways transcutaneous drug delivery has been the subject of debate for decades. From concrete stained of lipid synthesis or catabolism. Since the epidermis exhibits an extensive lipid metabolism necessary spots on skin surface during iontophoresis of charged dyes and high current density around these for the establishment of the barrier function, we have examined the role of PPAR-01α activation spots, ‘‘pores’’ or ‘‘shunt’’ have been denoted to a local, high conductance pathway. However, the in this process. Living skin equivalents were treated with Wy 14,653, a selective PPAR-01α physiological nature of such structure is not clear and has only being postulated to be related to ligand. The synthesis of membrane coating granules (MCGs) was greatly enhances. Also, the the skin appendages. overall stratum corneum neutral lipid content assessed by Oil red O staining was increased. A Recently, we anatomically studied pathway of sweat glands in human skin by using methylene detailed analysis of the lipid species present in the reconstructed epidermis showed that PPAR- blue. Full-thick skin was excised from cadaver within 12 h postmortem and prepared by removing 01α activation increased the synthesis of ceramides and cholesterol derivatives, thought to be fat tissue. It was mounted on an Ussing chamber with epidermal and dermal sides facing the essential structural components of the permeability lipid barrier. Furthermore, activation of donor and receiving compartments, respectively. The donor compartment was ﬁlled with 1% of PPAR-01α led to increased mRNA expression of several key enzymes of ceramide and methylene blue in PBS solution and the receiving compartment with only PBS. A direct current cholesterol metabolism. of 100 µA per cm2 was delivered across the skin disc with the donor compartment as the anode. Altogether, these results show that PPAR-01α is a key transcription factor involved in the control After 2 h, the solution in the receiving compartment was light blue in color. Interestingly, when of the epidermal lipid barrier. the skin disc was carefully hand-dissected under a stereo microscope, the sweat glands, and only the sweat glands showed dark blue color whereas the rest of dermis was colorless. It is necessary to point out that methylene blue did not stain the wall cells of sweat glands and the blue color was washable. High concentration of dye diffused into the sweat gland would have made them visible. These experiments clearly demonstrate that sweat glands function as a signiﬁcant shunt pathway to the stratum corneum.

497 498 Omega-Hydroxy Ceramides in Lipid-Bound Envelope (LBE) Formation The Effect of Methoxsalen Dose on UVA-Induced Erythema M. Behne, Y. Uchida, T. Seki, P. M. Elias and W. M. Holleran S. Ibbotson and P. Farr Department of Dermatology, UCSF and Dermatology Service, VA Medical Center, San Franci- Dermatology Department, Royal Victoria Infirmary, Newcastle-on-Tyne, UK sco, California Methoxsalen (microcrystalline preparation) is given at a standard dose of 0.6 mg per kg or 25 mg Omega-hydroxy ceramides (01ω-OH-Cer) are the predominant lipid species that are covalently per m2 in oral PUVA therapy but there is great variation in serum psoralen concentration between attached to cornified envelope (CE) proteins to form the lipid-bound envelope (LBE), while their patients, and therefore in degree of photosensitivity achieved. In patients who have a high UVA acylated derivatives are major components of the extracellular lamellae responsible for barrier minimal phototoxic dose (MPD), an increase in psoralen dose may be considered, likewise a dose function. Although the major lipid constituents of the LBE are known (through studies by Wertz reduction may be appropriate in highly photosensitive patients. Limited information is available et al.), the steps that lead to formation of this unique and key epidermal structure have not been on the erythemal effect of changing psoralen dose (Ljunggren et al, J Invest Dermatol 1981; 76:73). delineated. We present evidence here that formation of the LBE proceeds in a stepwise manner. We studied in 15 subjects the effect of varying the methoxsalen dose over a clinically relevant 01ω-Hydroxylation of very-long chain fatty acids (FA) is the first committed step in this process, range. Two hours after methoxsalen ingestion, serum concentration was measured (HPLC, P. followed by the condensation of the 01ω-OH-FA with sphingoid base to form 01ω-OH-Cer. Barnfield, St Thomas’ Hospital, London) and the subjects phototested at 350 Ϯ 30 nm (0.45–14 The 01ω-hydroxylation is catalyzed by a specific cytochrome p450-dependent enzyme (i.e. J per cm2). The MPD at 72 h was recorded and intensity of erythema measured with a reflectance CYP4A), inhibitable by amino-benzotriazol (ABT). Expression of CYP4A increases with differenti- instrument. Each subject was tested on three occasions using doses of 25 mg per m2 (conventional ation both in cultured human keratinocytes and in intact murine and human epidermis, consistent dose; CD) or CD Ϯ 10 mg. Median serum psoralen concentration increased from 85 to 143–239 with increased production and content of 01ω-OH-Cer in the outer epidermis. Topical applications ng per mL with dose increases from CD-10 mg to CD and CD ϩ 10 mg, respectively (P Ͻ ω of ABT result not only in a marked decrease in epidermal 01 -OH-Cer content, but also in a 0.05). Median MPD and D0.025 (the objective equivalent of the MPD, derived from the dose– significant reduction in covalently bound 01ω-OH-Cer and morphologically detectable LBE. response curve), were significantly reduced with increasing methoxsalen dose from CD-10 mg β 2 2 2 Since both 01 -glucocerebrosidase-deficient Gaucher mice, and pro-saposin-deficient animals (MPD 1.7 J per cm ;D0.025 2.8 J per cm ) to CD (1.2 and 1.4 J per cm ) and CD ϩ 10 mg (0.9 produce an LBE, highly enriched in 01ω-OH-glucosyl Cer (01ω-OH-GlcCer) (presented and 1.0 J per cm2)(PϽ0.01). The psoralen dose had no significant effect on the slope of the elsewhere at this meeting), covalent attachment of glucosylated-01ω-OH Cer to CE proteins dose–response curve. Thus, changing the dose of methoxsalen over a narrow therapeutic range appears to be the second step in LBE formation. Together, these results reveal the following likely significantly altered threshold erythema but not the rate of increase of erythema. sequence for normal LBE formation: (i) production of 01ω-OH-Cer; (ii) glucosylation to 01ω- OH-GlcCer; (iii) covalent attachment of 01ω-OH-GlcCer to CE proteins; followed by (iv) subsequent deglucosylation. However, since other nonglucosylated sources of 01ω-OH-Cer could exist in the epidermis, other mechanisms for LBE formation may be operative, as well 606 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

499 500 The SCID-Hu Xenogeneic Transplantation Model Allows Screening of Antipsoriatic Drugs Systemic Photodynamic Therapy with Verteporfin and Polychromatic Light is a Safe and Effective W. -H. Boehncke, M. Kock,* K. Hardt-Weinelt and M. Wolter Treatment for Psoriasis Department of Dermatology and *Animal Research Facility, University of Frankfurt, Germany T. Elshorst-Schmidt, A. Chan,* R. Kaufmann and W. -H. Boehncke Although there are numerous animal models for psoriasis most of them do not provide the full Department of Dermatology, University of Frankfurt, Germany, and *QLT Phototherapeutics complexity of this polygeneic disease. The SCID-hu xenogeneic transplantation model has Inc., Vancouver, Canada successfully been used in the analysis of the pathogenesis of psoriasis. Photodynamic therapy (PDT) has been reported to be effective in psoriasis. For routine application In order to investigate the applicability of this model for the purpose of screening of antipsoriatic in treating moderate to severe psoriasis patients systemic application of a photosensitizer and use therapies we transplanted lesional skin from three patients with chronic plaque-stage psoriasis onto of a polychromatic red light source that can irradiate a large bodysurface-area may have added female C.B17 SCID mice. Full-thickness skin donated by each patient was dissected into 12 grafts advantages. We performed an open nonrandornized phase I/II study treating 16 patients with and transplanted onto the back of the mice, each mouse carrying one graft. Four weeks later the chronic plaque-stage psoriasis in weekly intervals combining i.v. application of verteporfin mice underwent treatment for a period of another 4 wk receiving one of the following protocols: (benzoporphyrin derivate; 0.2 mg per kg) and subsequent irradiation with 600–700 nm light Daily s.c. injections with PBS served as negative controls; dexamethasone (0.2 mg per kg body (Waldmann PDT 1200, 60 J per cm2) for 5 wk. In five cases the study had to be terminated weight) administered once daily orally and BAY X 1005, an inhibitor of leukotriene synthesis, prematurely (3x thrmobophlebitis, lx lack of compliance, lx intercurrent not study-related given twice daily per os at a dose of 1 or 5 mg per kg body weight, respectively, were given as infection), 11 patients completed the study protocol. All patients exhibited partial responses as antipsoriatic treatments. Each group consisted of three mice. Thereafter, the mice were sacrificed, early as 1 wk after the initial treatment. The erythema had the tendency to fade before the and the grafts were analyzed by means of morphometry and histology. elevation of the lesions decreased suggesting that anti-inflammatory effects manifest first during PBS treated grafts retained all characteristics of lesional psoriatic skin. Dexamethasone treatment the course of the treatment. With the exception of raising triglyceride levels in one patient no resulted in a profound reduction of akanthosis and papillomatosis; parakeratosis was less obvious pathological changes occurred with regard to laboratory values throughout the study. Side-effects compared to PBS treated grafts resulting in a regular web-like structure of the corneal layer. The comprised a mild burning sensation at the treatment sites during the period of irradiation. One granular layer was completely re-established. Moreover, the inflammatory infiltrate was reduced, patient encountered symmetric swelling of both forearms within a day after the last treatment; and Munros´ abscesses could not be detected. BAY X 1005 reduced akanthosis, but all other this episode most likely was caused by exposure to natural day light during a weekend holiday characteristics of lesional psoriatic skin persisted. trip. The symptoms cleared without any persistent signs. We conclude that PDT combining i.v. This report documents that the phenotype of lesional psoriatic skin is reversible in the SCID-hu application of verteporfin and subsequent irradiation with polychromatic red light is a safe and xenogeneic transplantation model. The results obtained in this system correlate well with the effective treatment in chronic plaque-stage psoriasis; this approach might have the potential to clinical experiences. develop into a valuable alternative to other photochemotherapies such as PUVA.

501 502 Rapid Relapse After Complete Remission of Metastasized Merkel Cell Carcinoma by High-Dose Porokeratosis of Mibelli Treated with Tazarotene Gel and Topical Corticosteroids Polychemotherapy and Autologous Bone Marrow Transplantation J. J. Hong and M. Saraf, V. Sulica V. Waldmann, A. Jaeckel, M. Deichmann, M. Bock, U. Hegenbart, H. Goldschmidt, W. Hartschuh Department of Dermatology, George Washington University Medical Center, Washington DC and H. Naeher Porokeratosis of Mibelli is an uncommon cutaneous disorder that is characterized by annular Departments of Dermatology and Hematology/Oncology, University of Heidelberg, Germany plaques surrounded by a thin keratotic wall. Several nonsurgical methods have been utilized in a Merkel cell carcinoma (MCC) is a rare tumor of the skin of neuroendocrine differentiation. therapeutic modality with varying degrees of success. We describe a case of classical Porokeratosis Prefered site of metastases are regional lymph nodes, but multiple visceral metastases can occur. of Mibelli (PM) treated with tazarotene gel 0.05%. Therapy of choice consists of surgery, eventually followed by radiation. Chemotherapy has been A 54-y-old white male presented with a 5-y history of a pruritic, nonhealing lesion on his left performed with a variety of regimens based largely on agents active in small-cell lung cancer, leg that had been increasing in size despite an 8-mo course of corticosteroid therapy. Physical however, rapid relapses are common. As MCC is rare, there is no study-based standard protocol examination was remarkable for a solitary 4 cm erythematous plaque on the left anterior lower for treatment of metastasized disease. We report a 59-y-old male caucasian patient with multiple leg. There is a distinct peripheral keratotic ridge and central atrophy. Histopathology reveals small systemic metastases of MCC. The primary tumor had been excised, multiple local and lymphatic mounds of parakeratosis with minimal disorganization of the epithelium and a slight decrease in recurrences were treated by surgery and/or radiation. After diagnosis of systemic metastases, the the granular cell layer. There is subjacent band-like lymphohistiocytic infiltrate and epidermal patient was treated with a polychemotherapy composed of etoposide 150 mg per m2, cisplatin 80 atrophy beside the parakeratotic mounds. Treatment was begun with tazarotene gel 0.05% bid in mg per m2, doxorubicine 50 mg per m2, 30 mg bleomycine on day 1 and etoposide 150 mg per addition to topical fluocinonide acetonate ointment. Marked clinical improvement was noted after m2 on day 2, given four times in 3-wk intervals. This therapy was followed by a high-dose 3 mo. Rebiopsy was planned after completion of four months of therapy. chemotherapy according to the PEI regimen (ifosfamide 2400 mg per m2, carboplatin 180 mg PM is a condition with a complex pathophysiology and mostly unsatisfactory treatment alternatives. per m2 and etoposide 300 mg per m2), and subsequent bone marrow stem cell transplantation. Its etiology is believed to be related to genetic susceptibility and an autosomal dominant mode of After 3 mo complete remission of metastases could be diagnosed, however, this remission lasted inheritance with variable penetrance. Males are affected more than females, and the inherited form only three further months. This is the first report on a total remission of metastatic Merkel cell of this condition has been reported in children. It has been theorized that PM is the result of a carcinoma after high-dose polychemotherapy and autologous bone marrow transplantation. This mutant clone of epidermal cells which are activated in response to a variety of triggering factors. therapy seems to be powerful but benefit is of short duration and therefore of palliative nature. Progression to squamous cell and basal cell carcinoma is of particular concern and the estimation of this risk varies between 6.8% and 11%. A wide selection of treatment alternatives have been attempted, with varying degrees of success. Tazarotene is a novel retinoid which is selective for the RAR beta and gamma receptors and has shown to modulate keratinization and proliferation patterns of keratinocytes. This report is the first of its kind showing the effects of tazarotene on classical PM.

503 504 Effects of Roxithromycin on the Growth of Neurofibroma-Derived Cultured Cells from Patients Enzymatic Characterization of Six Immortalized Human Keratinocyte Cell Lines, Comparison of Neurofibromatosis Type 1 with Primary Cultures S. Imakado, E. Ichikawa, T. Kawashima, Y. Kawachi and F. Otsuka J. Cotovio, M. Baur, P. Justine, R. Roguet and P. Catroux Department of Dermatology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, L’OREAL, Life Science Research, Aulnay sous Bois, France; NESTLE Research Center, Ibaraki, Japan Lausanne, Switzerland Neurofibromatosis type 1 (NF-1) is an autosomal-dominant inherited disorder, characterized by The aim of this study was to characterize some metabolizing and antioxydant enzymes expressed the presence of six or more cafe au lait spots, axillary freckles, multiple neurofibromas and Lisch in cultured human keratinocytes potentially used in in vitro pharmacotoxicological studies. We nodules. Genetic linkage studies have confirmed that the gene for NF-1 is located on chromosome measured the activity of some phase I [ethoxyresorufin-O-deethylase (EROD), ethoxycoumarin- 17 and the gene product, neurofibromin, has a function as a GTPase-activating protein. However, O-deethylase (ECOD), esterase] and phase II [glutathione-S-transferase (GST), UDP-glucuronyl- treatment methods of cutaneous lesions of NF-1 are limited, such as surgical excision of transferase (GT), sulfotransferase (ST) and DT-diaphorase (DTD)] drug metabolizing enzymes as neurofibromas and laser therapy for pigmented spots. To test the possibility that roxithromycin well as antioxidant enzymes [superoxide dismutase (SOD), glutathione peroxidase (GPx) glutathione may have a suppressive effect on the growth of cutaneous neurofibromas, we examined the effects reductase (GR) and catalase(Cat)] of six immortalized human keratinocytes (IHK). Comparison of roxithromycin on the growth of neurofibroma-derived cultured cells from patients of NF-1. was made with normal adult human keratinocytes (NHK) in culture. IHK models were Number of neurofibroma-derived cells from one NF-1 patient, which were cultured with 50 µg immortalized either spontaneously (NCTC2544, HaCaT), or by SV-40 T-Ag transfection (SVK14, per ml of roxithromycin for 4 d, decreased significantly 10%, when compared with those cultured DK3-NR, FK2-NR) or SV-40 T-Ag/HPV16 E6/E7 transfection (DK7-NR). Our results showed without it. Number of neurofibroma-derived cells from another NF-1 patient also decreased 8%, that IHK as well as NHK retain in vitro 3-MC inducible EROD (mainly supported by CYP1 A1) when cultured with 10 µg per ml of roxithromycin for 8 d. Roxithromycin also suppressed the and ECOD (predominantly 1A and 2B-dependent) activities. All IHK express esterase activity at TGF-01β1 induced growth stimulation of neurofibroma-derived cultured cells about 60%. We least equal to NHK or three times higher (DK7-NR and SVK14). Phase II enzymes catalytic are now trying to further elucidate how roxithromycin acts on the growth of neurofibroma- activities were detected in all models. As compared to NHK, GST activity in DK7-NR and derived cultured cells from patients of NF-1. HaCaT was similar, whereas NCTC2544 and SVK14 expressed a lower activity. ST activity in NHK was very weak (Ͻ0.02 pmol per min per mg protein) whereas cell lines exhibited a slightly higher activity (up to 7.6 for NCTC2544). GT was present in NHK and in IHK but with different activity levels. Indeed, 111 and 48 pmol per min per mg protein were found for HaCaT and DK7-NR, respectively, versus only 8 for NHK. FK2-NR, HaCaT and NCTC2544 cells showed very high DTD activities (316, 346 and 319 nmol per min per mg protein, respectively) versus 16 for NHK. Antioxidant enzymes were highly expressed. DK7-NR and FK2-NR showed a GPx activity two times higher than NHK (35 mU per mg). All Cat, GR and SOD activity levels in IHK, were at least as higher as in NHK. These results show that: Cultured NHK retain some drug metabolizing enzyme capabilities as well as antioxidant enzyme capabilities; IHK exhibit similar enzyme profiles than NHK, but with different activity levels. Therefore, the use of IHK as pharmacotoxicological tools should be considered. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 607

505 506 Effects of Nuclear Receptor Agonists on Repair of Photodamage in Mouse Skin Lipodermatosclerosis Treated with Colchicine and Pentoxifylline G. J. Gendimenico, V. J. Mack and J. A. Mezick J. Kwon and I. Aronson Johnson and Johnson Consumer Products Worldwide, Skillman, New Jersey Department of Dermatology, University of Illinois at Chicago, Chicago, Illinois Retinoids are well known for their ability to induce new connective tissue synthesis in Lipodermatosclerosis is a progressive disorder associated with venous insufficiency, most commonly photodamaged hairless mouse skin. Because these effects are mediated by retinoic acid nuclear of the lower leg, characterized by an acute inflammatory stage and a chronic fibrotic stage in receptors of the steroid/thyroid hormone family, we determined if other hormonal compounds which ulceration may occur. Therapeutic modalities consist mainly of compression and enhanced had photodamage repair activity. Hairless mice were exposed to gradually increasing doses (1–4.5 fibrinolysis (stanozolol). We treated five patients with the diagnosis of lipodermatosclerosis with MEDs) of UV from fluorescent tubes from weeks 1–5, then 4.5 MEDs from weeks 5–10. UV the combination of colchicine (1.2 mg daily) and pentoxifylline (1200 mg daily), in addition to exposure was stopped and then skin was topically treated with test agents, three times per week compression. All the patients experienced marked improvement with decreased pain, erythema, for up to 10 wk. Connective tissue repair zone depth was quantified by image analysis of edema, induration, and ulceration after 2–3 mo of therapy. The therapeutic effects of these two histological sections. The hormonal compounds that were evaluated included an androgen, drugs may be mediated by a variety of mechanisms, including inhibition of neutrophil migration estrogens, glucocorticoids, a progestin and thyroid hormone. Only estrogens (0.05% dose) 17- and degranulation, inhibition of expression of TNF01α receptors on the surfaces of macrophages 01β-estradiol (E2) and diethylstilbestrol were effective at stimulating connective tissue repair zones and endothelial cells, increased collagenase production, and decreased collagen deposition. The after 10 weeks of treatment. E2 caused dose-related repair activity, with 0.1% E2 having effects combination of colchicine and pentoxifylline is an alternative modality for this therapeutically similar to 0.03% all-trans-retinoic acid (RA). Like RA, E2 (0.1%) also caused epidermal hyperplasia challenging disorder. in photodamaged skin after 10 wk of treatment. Additionally, E2 at 0.01 and 0.1% caused an increase in skin-fold thickness in normal mice after only three consecutive, once daily topical treatments. The effects of estrogens described here are consistent with clinical reports of estrogens having beneficial effects in aged human skin

507 508 The Incidence and Significance of Cancellation and Non-attendance at a Dermatology Clinic Management of Molluscum Contagiosum in HIV-Infected Males with a Dual Combination N. Penneys and D. Glaser Therapy (1% Imiquimod Cream) and Zidovudine 250 mg. A Placebo-Controlled and Double- Department of Dermatology, Saint Louis University, St. Louis, Missouri Blind Study Same-day cancellation of appointments and nonattendance (NA) by patients can disrupt attempts T. A. . Syed,* Z. A. Qureshi and S. A. Ahmad to manage medical care delivery and leads to inefficient allocation of resources and lost revenue. *Department of Dermatology, University of California San Francisco, California, University of We measured same-day cancellation and NA in a university dermatology clinic. Wales College of Medicine, Department of Dermatology, Cardiff, UK, and Windsor High School, We tabulated clinic attendees, same-day cancellations, and NA for a 6-mo period. We examined Windsor, California distribution by gender, type of payer, and clinic visit type (initial versus follow-up). The purpose of this study was to evaluate the clinical efficacy and tolerability of 1% imiquimod 4876 patient visits were surveyed for this study. Total same-day cancellation rate was 8.3%. Females cream to cure HIV-infected patients who were already on oral medication of Zidovudine (250 canceled more frequently than males. Cancellation rates were similar for initial and follow-up mg). Fifty male patients aged between 18 and 60 y with confirmed HIV infection (both HIV-1 visits. The NA rate was 16.9% with the highest rates found in public aid categories (26%) and the and HIV-2) diagnosed by polymerase chain reaction and having concomitant Molluscum lowest, in commercial insurance programs (12.7%). NA rates did not vary by gender or visit type. contagiosum joined the study. The diagnosis of Molluscum contagiosum was enhanced both by The variance per clinic was significant and varied from 0% cancellation and NA to 100% histology and in situ hybridization using a biotinylated probe. Subjects were not allowed to use cancellation and NA for public aid categories. any oral or systemic medication during the trial period except 250 mg Zidovudin twice daily Same-day cancellation and clinic NA is a significant impediment to efficient fiscally sound three times a week. Patient were sequentially assigned to receive a precoded 25-g tube of trial management of a busy dermatology clinic. The risk of NA is assumed by physicians and seems to preparation (active or placebo). Patients self-administered the trial medication to their lesions at be correlated with payer type. The variance per clinic suggests that programmed overbooking will home, once daily for three consecutive days per week. The study was scheduled for four weeks lead to decreased quality of care on days with little cancellation and NA. of active treatment (max. 12 topical applications) and during that period patients were evaluated on a weekly basis, after that subjects were followed up on a monthly basis for 12 months. Five patients had atopic dermatitis. A clinically and histopathologically confirmed total elimination of lesion was considered as cured. By the end of the treatment findings indicated that 50% patients were cured, breaking the code revealed that 1% imiquimod cream had cured 84% patients and 85.9% (165 of 192) lesions. Placebo cleared 16% patients and 17.3% (29 of 168) lesions, P Ͻ 0.0001. 86% patients complained of no drug-related side-effects. Seven patients 14% predominantly in the active cream medication group experienced nonobjective mild headache, fever, itching or a combination of two or all with no dropout. Among cured patients four subjects relapse after 11 months. In conclusion, the study demonstrated that 1% imiquimod incorporated cream is tolerable and significantly more effective than placebo to cure Molluscum contagiosum, and the regimen can be considered as a reliable dual treatment modality to cure Molluscum contagiosum in HIV- positive patients.

509 510 SDZ ASD 732 Represents a Novel Type of Anti-Inflammatory Ascomycins Which do Not Bind Topical SDZ ASM 981 and Clobetasol-17-Propionate Show Similar Inhibition of the DNFB- to Macrophilin Induced Allergic Contact Dermatitis in Go¨ttingen Minipigs K. Baumann, P. Hiestand,* F. Kalthoff, J. Meingassner, W. Schuler,* H. Schuurman,* A. Stuetz J. Meingassner and G. Vana and M. Grassberger Novartis Research Institute, Vienna, Austria Novartis Research Institute, Vienna, Austria and *Novartis Pharma, Basle, Switzerland SDZ ASM 981 is a new anti-inflammatory ascomycin macrolactam. Since DNFB-induced allergic Pharmacological evaluation of large series of ascomycin derivatives has shown that affinity to contact dermatitis (ACD) in Go¨ttingen minipigs (GMP) has proven to be a predictive animal macrophilin, the cytosolic receptor of ascomycin/FK506/rapamycin, does not always correlate model for human acute ACD concerning clinical and immunohistological features of human acute with the anti-inflammatory activity in animal models of allergic contact dermatitis. Here we ACD, we investigated the activity of SDZ ASM 981 in this animal model. Comparison was made present data on SDZ ASD 732, an ascomycin derivative, that does not bind to macrophilin. SDZ with the activity of the ultrapotent corticosteroid clobetasol-17-propionate (CLO). ASD 732 inhibits auricular hypersensitivity reactions in mice sensitized to oxazolone after topical, Eight GMP were sensitized with 10% DNFB and challenged 2 weeks later with 1% DNFB. The subcutaneous (sc), or oral administration as assessed by inhibition of inflammatory pinnal swelling test sites were treated twice (0.5 and 6 h after challenge) either with SDZ ASM 981, CLO (both and influx of neutrophils (determined by myeloperoxidase activity in auricular tissue). This effect at 0.5% and 0.05%) or with the vehicle (ethanol/propylene glycol). Efficacy was assessed by was confirmed in models of DNFB-induced allergic contact dermatitis in rats (sc) and pigs (topical). clinical investigation, measurement of skin color and cutaneous blood flow, 24, 48 and 72 h after Anti-inflammatory activity was also observed in other models, not related to allergic contact the challenge. Sections of biopsies (collected 24 and 72 h after the challenge) were Giemsa-stained dermatitis, such as zymosan-induced pinnal inflammation (mice, guinea pigs; oral treatment) and or stained with a panel of mouse monoclonal antiswine antibodies for quantitative inflammatory reversed passive cutaneous Arthus reaction (rats; oral treatment). SDZ ASD 732 does not bind to cell analysis. rh-macrophilin-12 and is inactive in a series of in vitro assays for T cell activation (T cell receptor In comparison with vehicle-treated contralateral sites, SDZ ASM 981 at 0.5 and 0.05% inhibited mediated cytokine release and proliferation, transcription of a reporter gene that is under the erythema and induration by 60% and 40%, respectively, when inflammation peaked (24 h after control of the human IL-2 promoter, mixed lymphocyte reaction). SDZ ASD 732 does not affect challenge). Histopathology mirrored the in-life findings. Microscopic findings in sections of test the primary immune response in mice (plaque forming cell assay, sc application). Lack of sites treated with 0.5% SDZ ASM 981 were almost comparable at 72 h with those of uninvolved immunosuppressive activity was confirmed in a series of animal models, such as localized graft- skin. Cell counts in the dermis of 0.5 and 0.05% SDZ ASM 981-treated sites revealed a reduction versus-host reaction (rat, sc), heterotopic heart allotransplantation (mouse, sc, Alzet pump), and in CD45ϩ cells by 70% and 58% and CD2ϩcells were reduced by 63% and 61% 24 h after orthotopic kidney allotransplantation (rat, sc). SDZ ASD 732 thus represents a completely novel challenge. At 72 h CD25ϩ (IL2 Rϩ) cells were reduced by 78% and 70% and CD8ϩ cells by 85% type of anti-inflammatory ascomycin derivatives. and 62%. The responses of test sites treated with CLO did not differ significantly from SDZ ASM 981-treated sites. These results indicate comparable efficacy of SDZ ASM 981 and the ultrapotent corticosteroid clobetasol-17-propionate at 0.5% and 0.05% in this animal model. 608 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

511 512 SDZ ASM 981 Inhibits Anti-IgE Stimulated Mediator Release in Human Dermal Mast Cells Propylene Glycol Alters Dermatologic Corticosteroid Activity In Asian Versus Caucasian Skin T. Zuberbier, S. Chong, S. Guhl, P. Welker, B. Henz and M. Grassberger* L. Pershing, K. Petelenz and J. Corlett Department of Dermatology, Charite´-Virchow Clinic, Humboldt University Berlin, Germany; Department of Dermatology, University of Utah, Salt Lake City, Utah *Novartis Research Institute, Vienna,. Austria Previous work in our laboratory has demonstrated that dermatologic corticosteroid (DC) activity Mast cells are well characterized effector cells in allergic and immunological reactions, but only in skin, as measured by reflectance colorimetery a* scale, is influenced by vehicle composition, little is known about the mechanisms modulating their mediator release. Corticosteroids and steroid potency and is dose responsive: increasing the DC application duration increases the area- Cyclosporin A which efficiently inhibit mast cell mediator release are limited in their use due to under-the-skin blanching response effect-over time curve. Population-fitting the dose response side-effects and in case of Cyclosporin A also the disadvantage of not being topically applicable. data to ascertain the maximal DC activity (Emax) and the dose required to produce 50% of the Recently the ascomycin macrolactam derivative SDZ ASM 981 has been developed for the maximal response (ED50) can be used to compare steroid potency and bioavailability in humans. treatment of immunologically mediated skin diseases. In this study we have investigated the The purpose of this study was evaluate the influence of ethnicity on Emax and ED50 in gender capacity of this novel drug to inhibit mediator release from human skin mast cells.Human dermal and age-matched subjects (n ϭ 4–12 in each race group) with eight different TC products ranging mast cells were isolated from normal skin (from cosmetic surgery) by enzymatic dispersion. Cells in potency (Stoughton potency I–VI), drug and/or vehicle type (ointment, cream, gel, and lotion). were passively sensitzed with IgE overnight and preincubated with SDZ ASM 981 (10 nM – 5 In general, DC potency is associated with a larger negative Emax and a shorter ED50 value. µM) for 1 h prior to 20 min stimulation with anti-IgE (1000, 2000, 4000 IU per ml). Histamine However, Asian subjects demonstrated a 6–10X increase in ED50, yet similar Emax values, was determined spectrofluorometrically, tryptase activity photometrically after cleavage of the compared to Caucasians, with all evaluated DC products containing propylene glycol, independent specific substrate 2-gly pro-arg-p-nitroanilide in the supernatants.Values are presented as percentage of the vehicle type or steroid potency. DC formulations without propylene glycol produced of total cellular mediator contents. Pre-incubation with SDZ ASM 981 inhibited both anti-IgE similar Emax and ED50 values in both ethnic groups. Thus, vehicle composition of the stimulated histamine and tryptase release whereas spontaneous mediator release was not affected. DC product has a dramatic effect on the resulting pharmacodynamic activity in skin as a function Maximum inhibitory activity was seen with 500 nM SDZ ASM 981 with histamine release being of ethnicity. Increasing the ED50, while maintaining a similar Emax, produces a reconstructed reduced from 12.3% Ϯ 2.6% (buffer treated controls) to 2.7% Ϯ 0.6% (2000 IU anti-IgE, n ϭ dose response curve in which 10X longer application durations are required to achieve the same 10, P Ͻ 0.01). For comparison, maximum inhibition achieved with Cyclosporin A (100 nM) was maximal SBR activity in human skin in vivo. Increased ED50 values for the Asian subjects were a reduction to 7.0% Ϯ 3.1% and dexamethasone (1 µM) 7.5% Ϯ 2.9% which was similar with not the result of DC product n˜induced erythema. Despite dif-ferences in ED50 between the that seen with a 10-nM concentration of SDZ ASM 981. Comparable results were also seen on ethnic groups, the relative potency ranking among the DC products was similar in the Asian and measuring tryptase release, 500 nM SDZ ASM 981 inducing a mean inhibition of 76% compared Caucasian groups. Nonetheless, the increased dose required to achieve the maximal inherent to untreated controls (stimulation with 2000 IU anti IgE, n ϭ 3). In summary, these data show activity of a DC product containing propylene glycol decreases the overall effectiveness in Asian for the first time the inhibitory capacity of SDZ ASM 981 on mediator release in human dermal skin. Data suggest that DC products containing propylene glycol likely provide suboptimal therapy mast cells with a potency far exceeding both Cyclosporin A and dexamethasone underlining its for Asian subjects. potential in the treatment of IgE mediated diseases

513 514 Molecular Basis of Glucocorticoid-Induced Skin Atrophy: Topical Glucorticoid Decreases Both Capsaicin:Therapeutic Activities on Itch by Depleting Sensory Neuropeptides Fibers in the Skin Collagen Synthesis And Corresponding mRNA Level in Human Skin In Vivo 1. Ghersetich, B. Bianchi and T. Lotti A. Oikarinen, K-M. Haapasaari, M. Sutinen and K. Tasanen Department of Dermatology, University of Florence, Italy Departments of Dermatology and Dentistry, University of Oulu, Finland. Neuropeptides are a heterogeneous group of more than 50 molecules that play a role in various The effect of topical betamethasone-17-valerate (BM) on collagen propeptide levels, collagen cutaneous functions and disease. In the skin neuropeptides are synthesized locxally (i.e. in mRNA, lysyl oxidase mRNA and matrix metalloproteinase-1 (MMP-1) and matrix metallo- keratinocytes and in endothelial cells) and are transported by nerve fibers or immune cells. Specific proteinase-2 (MMP-2) mRNA levels were studied in human skin. Three days treatment of healthy receptors and binding sites for neuropeptides have been described in different cell lines in the skin skin with BM caused a 70%–80% decrease in type I and III collagen propeptides in suction blister (keratinocytes, endothelial cells, immune cells, fibroblasts). Many different biologic actions of fluid. A corresponding decrease was observed in type I collagen mRNA when assayed either by neuropeptides have been demonstrated Some neuropeptides (Substance P and related tachikinins slot blot hybridization or quantitative PCR method, indicating that the decrease of collagen and Calcitonin gene related peptide) are present in small diameter priary afferents that most likely synthesis after topical glucocorticoid is mostly due to reduced level of corresponding mRNA. belong to the C-fiber group and are mainly involved in pain and pruritus transmission and in Lysyl oxidase, which is an important enzyme catalyzing cross-linking of collagen chains, and process of tissue repair.Capsaicin excites C-fibers and releases tachykinins and CGRP; after repeated MMP-1 and MMP-2 mRNA were not declined in the same samples. application the short-term response is followed by tachphylaxis, which is thought to reflect the This indicates that in vivo glucocorticoids modulate variably genes involved in connective tissue depletion of Substance P and Calcitonin gene Related Peptide from the C-fibers.We evaluated synthesis and degradation. Furthermore, our study gives conclusive molecular basis of glucocorticoid the symptom pruritus before and after the application thrice a day of a 0.01% capsaicin cream on induced dermal atrophy, which is due to the decrease of functional collagen mRNA in the 72 patients affected by different dermatoses. In 12 of 72 patients we evaluated SP- and CGRP- skin in vivo. fibers by direct immunofluorescence before and after the treatment. Our results have shown that capsaicin cream clinically reduces pruritus and determines the reduction or disappearance of SP and CCRP fibers by immunofluorescence. (Lotti T. et al. J Am Acad Dermatol 1995; 33:482–496; Hylden JLK et al. Brain Res 1981; 217:212–219.)

515 516 01α-Melanocyte Stimulating Hormone: a New Potent Anti-inflammatory Agent Pharmacokinetics of LFA3TIP (Amevive) in Chronic Plaque Psoriasis Patients During Repeated B. Bianchi, I. Ghersetich and T. Lotti Once-Weekly Intravenous Administration Department of Dermatology, University of Florence, Florence, Italy M. Rogge, C. Ellis, G. Krueger, M. Cooney, G. Winkler, D. Magilavy and K. Sweeney 01‰[capsigma]0001[captau]α-Melanocyte Stimulating Hormone 01(α-MSH) is a neuroimmuno- Biogen, Inc., Cambridge, Massachusetts; University of Michigan, Ann Arbor, Michigan; University modulating peptide derived from the precursor hormone proopiomelanocortin; it is detected in of Utah, Salt Lake City, Utah; and University of Connecticut, Farmington, Conneticut many human tissues including the skin. 01α-MSH has been shown to have many functions, i.e. LFA3TIP (being developed by Biogen, Inc., under the trademark Amevive), a recombinant 115 it induces human melanogenesis and lipolysis, modulates the immune system response and has a kDa dimeric glycosylated protein consisting of the first extracellular domain of human LFA-3 potent anti-inflammatory and antipyretic activity fused to the hinge and CH2 and CH3 sequences of human IgG1, modulates immune responses In particular, central 01α-MSH administration has been shown to inhibit cutaneous inflammation through interaction with the CD2 receptor. The pharmacokinetics (PK) of LFA3TIP during induced by application of topical irritants and intradermal injection of cytokines probabily through repeated once-weekly intravenous (IV) administration in 24 patients with chronic plaque psoriasis descending pathways in nude mice. 01α-MSH exerts potent anti-inflammatory influences via (CPP) are presented. Serum drug concentration data were analyzed by population PK methods. direct actions on peripheral cells, such as monocytes/macrophages and neutrophils, reducing the Demographic variables were evaluated for significance in the estimation of the PK parameters. release of proinflammatory cytokines TNF-01α and IL-1. Moreover, 01α-MSH seems to modulate Doses ranged from 0.005 to 0.075 mg per kg per wk (5–7 patients per dose group). Blood for inflammation by influencing the expression of the adhesion molecules E-selectin and VCAM-1, PK analysis was drawn throughout the 8-wk dosing period and up to 7 wk following the last that is markedly affected by the presence of the melanotropic peptide. Studies performed on dose. A 2-compartment model best described the data. Although relatively constant among all animal models have shown that the cutaneous application of 01α-MSH may represent a useful patients, serum drug clearance was dependent on weight and age. Distribution volumes did not approach in the treatment of different inflammatory skin diseases. Recent data from our and other correlate with age, weight, height or body mass index. An insufficient number of females and groups show that with properly vehiculed application 01α-MSH exerts a potent anti-inflammatory non-Caucasians in the study population precluded gender and race from the demographic analysis. action on human skin. Overall, mean serum drug clearance was ~0.18 ml per h per kg. The central and peripheral (Teofoli P, Guardello V, Lotti T, Panconesi E. Int J Immunopathol Pharmacol 1997; 10:69–71; distribution volumes were ~4.3 liters and ~2.2 liters, respectively. The elimination half-life was Teofoli P, Motoki K, Lotti T, Uitto J, Mauviel A. Exp Dermatol 1997; 6:111–115; Lotti T, 890 h (~5 wk). Residual error ranged from 18% to 32%. These results indicate that once-weekly Hautmann G, Panconesi E. J Am Acad Dermatol 1995; 33: 482–496; Luger TA, Lotti T. J Eur administration of LFA3TIP in CPP patients is predictable and that it may not be necessary to Acad Dermatol Venereol 1998; 10:207–211.) adjust dose based on age or weight. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 609

517 518 Interleukin-12 Induces Lesion Regression and Cytotoxic T-Cell Responses in Cutaneous T- Pharmacodynamic Effects of LFA3TIP (Amevive) in Patients with Chronic Plaque Psoriasis (CPP): Cell Lymphoma Selective Modulation of CD45ROϩ Lymphocytes A. Rook, E. Vonderheid, E. Yoo, D. Kao, R. Elenitsas, M. Sherman, R. Shane, S. Lessin and D. Magilavy, T. Mant, P. Norman, G. Krueger, C. Griffiths, C. Ellis, J. Barker, G. Winkler and G. Wood M. Rogge University of Pennsylvania, Allegheny University, Case Western Reserve Medical Center, and Biogen, Inc., Cambridge, MA, UMDS-Guy’s St. Thomas Hospitals, University of Utah, University Genetics Institute, Philadelphia, Pennsylvania, Cleveland, Ohio, and Cambridge, Massachusetts of Manchester, and University of Michigan Progression of cutaneous T-cell lymphoma (CTCL) is associated with profound defects in cell- LFA3TIP, a recombinant 115 kDa dimeric glycosylated protein which consists of the first mediated immunity (CMI) and depressed production of cytokines which support CMI. Because extracellular domain of human LFA-3 fused to the hinge and CH2 and CH3 sequences of human we have observed marked defects in interleukin 12 (IL-12) production in CTCL, and since IL- IgG1, has been designed to modulate immune responses through interaction with the CD2 12 is critical for antitumor cytotoxic T-cell responses, we initiated a phase I dose escalation trial receptor. Based upon the in vitro effects of LFA3TIP on T cell function and its therapeutic potential with recombinant IL-12 where patients received either 50, 100 or 300 ng per kg IL-12 twice in CPP, tolerability, pharmacokinetics, and biologic activity of LFA3TIP were evaluated in a single weekly subcutaneously or intralesionally for up to 24 wk. Ten patients have been entered: five dose, dose-escalation trial in two multidose dose-escalation trials in patients with CPP. In the first with extensive plaque, three with Sezary syndrome (SS) and two with extensive tumors with large psoriasis trial, 24 patients received eight once-weekly intravenous injections of LFA3TIP at 0.005 cell transformation. One patient with SS dropped out after 1 wk for personal reasons. Subcutaneous mg per kg, 0.025 mg per kg, 0.050 mg per kg, or 0.075 mg per kg. In the second psoriasis study, dosing resulted in complete responses (CR) in two of five plaque and partial responses in two of 18 patients received a total of two IV injections of LFA3TIP once every four weeks at 0.05 mg five plaque and one of two SS (overall response rate CR ϩ PR, five of nine, 56%). A minor per kg, 0.10 mg per kg, or 0.15 mg per kg. LFA3TIP was well tolerated with no drug-related response also occurred in one of five plaque patients. Intralesional dosing resulted in individual serious adverse events reported. A transient reduction from preinfusion levels in the absolute plaque, erythema or tumor regression in eight of nine patients. Biopsy of regressing lesions revealed number of peripheral lymphocytes was observed in all subjects who received LFA3TIP. This a significant decrease in the density of the infiltrate in all cases and complete resolution of the reduction was noted in total, CD2ϩ, CD4ϩ, and CD8ϩ lymphocytes but not in CD19ϩ infiltrate among those with clinical lesion resolution. An increase in numbers of CD8 positive lymphocytes. The degree and duration of reduction from baseline levels correlated directly with and/or TIA-1 positive T-cells were observed on immunohistochemical analysis of skin biopsies the dose of LFA3TIP. A selective decrease of high fluorescence intensity staining CD2ϩ cells in obtained from regressing skin lesions. Adverse effects of IL-12 on this regimen were minor and both the CD4 and CD8 subpopulations was observed by flow cytometric analysis. A greater limited and included low grade fever and headache. One patient discontinued IL-12 at week 6 reduction in CD2ϩ cells with memory/effector phenotypes (CD4ϩ-CD45ROϩ and CD8ϩ- due to depression. These results suggest that IL-12 may augment antitumor cytotoxic T-cell CD45ROϩ) than with naı¨ve phenotypes (CD4ϩ-CD45RAϩ and CD8ϩ-CD45RAϩ) was also responses and may represent a potent and well tolerated therapeutic agent for CTCL. observed. Selectivity for CD45ROϩ cells over CD45RAϩ cells may be related to increased expression of CD2 on CD45RO cells. These results indicate that LFA3TIP is well tolerated and shows unique PD properties in CPP, including selectivity for memory/effector T cells.

519 520 The Assessment of Seasonal Affective Disorder in the Tanning Bed Population; A Survey of The Reactive Oxygen Species (ROS) Regulate the Gene Expressions Involved in the Biosynthesis Tanning Bed Users and Biodegradation of Collagens in Three-Dimensional Culture of Normal Human Dermal S. Gee and R. Dufresne Fibroblasts Department of Dermatology, Wayne State University, Detroit, Michigan; Department of Dermato- K. K. Zaw, Y. Yokoyama, O. Ishikawa, and Y. Miyachi* logy, Brown University School of Medicine, Providence, Rhode Island Department of Dermatology, Gunma University School of Medicine, Japan; *Department of Tanning bed users were evaluated for symptoms of Seasonal Affective Disorder (SAD) to determine Dermatology, Kyoto University Graduate School of Medicine, Japan if there is a correlation between these symptoms, tanning bed use, and perceptions of well-being. Extracellular matrix changes in photodamaged skin may be the consequence of the alteration in Fifty-two tanning bed patrons were asked to fill out a questionnaire to determine the presence biosynthesis and degradation of dermal connective tissue components. Ultraviolet irradiation to and extent of SAD symptoms. Questionnaires were adapted from the Seasonal Pattern Assessment cutaneous cells can generate ROS that are known to play important roles in regulating the gene Questionnaire (SPAQ) of Rosenthal, Brandt and Wehr. Respondents were asked to determine expression of extracellular matrices by dermal fibroblasts. Normal dermal fibroblasts grown in the their pattern of seasonality by indicating the specific months they feel best and worst during the three-dimensional culture supplemented with L-ascorbic acid 2-phosphate were exposed to ROS year and to determine their degree of seasonality by indicating degree of change in sleep length, generated by xanthine-xanthine oxidase system. Total RNA was extracted and subjected to social activity, mood, weight, appetite and energy level, and quantitating weight fluctuations and northern blot analysis using probes for human interstitial collagenase (MMP-1), 72 kDa type IV α α hours of sleep during the year. Degree of seasonality was scored according to Rosenthal’s severity collagenase (MMP-2), tissue inhibitor of metalloproteinase 1 (TIMP-1), type 01 1(I) and 01 1(III) scale included in the questionnaire. The mean score of our respondents is 14, which indicates our collagens. ROS elevated the expression of MMP-1, MMP-2 and TIMP-1 mRNA at 48 and 72 α α participants suffer from moderate to extreme symptoms of SAD. All tanning bed patrons in the h after exposure. In contrast, it suppressed both the type 01 1(I) and 01 1(III) collagen mRNA study felt that there were particular months which effected them more negatively than others. at 48 and 72 h after exposure. Catalase completeyl blocked the ROS-induced alteration of MMP- α α These respondents showed primary winter seasonal patterns, feeling worse in the winter months 1,TIMP-1, type 01 1(I) and 01 1(III) collagen mRNA expression. It, however, did not block of December, January and February. Frequency of tanning bed use increased during these months ROS-induced MMP-2 mRNA level. SOD had no effect on the ROS-induced changes. Gelatin among our respondents. All participants reported relief from their SAD symptoms with the use zymography demonsterated the increased conversion from a latent form to an active form after of the tanning bed. These data suggest that this tanning bed population suffers from SAD and exposure to ROS. Neither catalase nor SOD blocked the increased conversion of MMP-2. The patronized tanning salons in an effort to ‘‘self -medicate’’ and feel less depressed during the results suggest that loss of collagen fibers in photodamaged skin are partly due to the elevated winter months. collagenolytic activity and the suppressed biosynthesis of collagens by dermal fibroblasts exposed to ROS.

521 522 In Vitro Evidence of Efficient Biostimulatory Effects of Linear Polarized Near Infrared Light Dendritic Cells (DC) Irradiated with Low-Dose UVB are Impaired in their Allostimulatory Irradiations on the Acceleration of Cutaneous Wound Healing Capacity, but do Not Induce Specific Tolerance in Allogeneic T Cells K-I. Toda, K. Danno,* S. Imamura† and Y. Miyachi J. P. Tesmann, R. W. Denfeld, T. Wachter, E. Scho¨pf and J. C. Simon Department of Dermatology, *Graduate School of Medicine, Shiga Medical College, †Matsue Department of Dermatology, University of Freiburg, Freiburg, Germany City Hospital and Kyoto University, Japan Recently, we have shown low-dose UVBR to perturb the expression of CD80 and CD86 on We previously reported that nonpolarized near infrared light (NPNIR) irradiations significantly Langerhans cells, thereby inhibiting their capacity to induce allo- and antigen-specific T enhanced cutaneous wound healing. Further, previous studies indicated that the irradiations of cell responses. Furthermore, we have demonstrated UVBR to convert Langerhans cells from linear polarized near-infrared light (PNIR) accelerated the healing rate in mammals. In these immunogenic to tolerogenic APC. DC have attained special interest for immunotherapeutic context, the cell biological effects of NPNIR irradiations on the cultured cells as well as an in vitro applications, i.e. autoimmunity, since they can be generated in large numbers and have similar wound model were studied and those effects of NPNIR and PNIR were comparatively analysed. effector functions compared to Langerhans cells. PNIR and/or NPNIR system [Super Liser (SL), HA550, Tokyo Iken, Tokyo, Japan] were used Therefore, we questioned whether low-dose UVBR of DC alters their allostimulatory capacity, as the light source. Normal human cultured keratinocytes (KC), fibroblasts (FB) and microvascular thereby inducing tolerance in T cells. To address this issue, primary one-way mixed leukocyte endothelial cells (EC) were irradiated by SL on ice. The output of NPNIR at the lens unit was reaction (MLR) was performed using bone-marrow derived C57/BL6 DC and BALB/C 2200 mW per cm2 with the focused diameter of 10 cm. That of PNIR at the lens was 1800 mW lymphnode cells. First, we found low-dose UVB-DC to dose-dependently (50–200 J per m2) per cm2 with the focused diameter of 1 cm. Irradiated doses per day were ranged from 0 to 28.6 inhibit T cell proliferation. In addition, supernatants harvested from MLR with UVB-DC showed J per cm2, monitored by the laser power meter, PM-313 (Japan Science Engineering, Tokyo) 3 reduced levels of Th1 cytokines, IL-2 and IFN-01γ, compared to MLR with sham-irradiated DC cm under the lens. Irradiations were continued for 5 d over a 24-h period. The cell growth and while IL-4 and IL-10 levels remained unaffected. This suggests a preferential downregulation of migration were examined by MTT assay and phogokinetic assay, respectively. MTT assays showed Th1 responses by UVB-DC. FACS-analysis of UVB-DC revealed no changes in surface expression that both NPNIR and PNIR irradiations on KC, FB and EC cells comparatively incresed of MHC class II (Iab), the costimulatory molecules CD40, CD80 and CD86, as well as the percentage optical density of the treated cells in a dose dependent manner, to 165%, 145% and adhesion molecules CD11c and CD54. Importantly, we could exclude any effects of UVBR on 228% of nonirradiated controls, respectively, at the irradiated doses of 28.6 J per cm2. Phagokinetic DC viability as determined by propidium iodide staining. To test tolerance induction, T cells assays on the fibronectin coated culture dishes showed that the migration index of the NPNIR- harvested from primary MLR were restimulated with either unirradiated C57/BL6 DC (Iab), irradiated EC was 1.5 times larger than that of nonirradiated controls and that the index of PNIR- C3H/Hen DC (Iak) or IL-2 (100 U per ml). Interestingly, even 4 h of UVB-DC/T cell coculture irradiated cells was 3.5 times larger than the controls. In the skin explant culture, the migrated in primary MLR induced partial inhibition of T cell proliferation upon restimulation. However, areas of the treated epidermal sheets were also 2.1 times or 2.8 times larger by NPNIR or PNIR T cells showed similar unresponsiveness to restimulation with IL-2 and unirradiated DC irrespective irradiations than those of controls, respectively. These results indicate that near infrared light of their haplotype. irradiations directly activate skin cells including KC, FB anf EC to accelerate the wound healing In conclusion, we show that low-dose UVBR alters the allostimulatory capacity of DC but does and the effectiveness of the irradiations was more prominent in PNIR than in NPNIR not allow tolerance induction in T cells to alloantigen. These results suggest that DC respond differently to UVBR compared to LC. 610 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

523 524 Low-Dose UVB-Irradiated Dendritic Cells (DC) Induce Apoptosis in Allogeneic T Cells by a Low-Dose UVB Radiation Induced Suppression of Dendritic Cell Antigen Presenting Function Fas/FasL Independent Pathway is Not Related to Cyclobutane Pyrimidine Dimer (CPD) Formation R. W. Denfeld, T. Wachter, J. P. Tesmann, E. Scho¨pf and J. C. Simon H. C. Dittmar, U. S. Haaga, D. B. Yarosh,* O. Nikaido,† E. Scho¨pf and J. C. Simon Department of Dermatology, University of Freiburg, Freiburg, Germany Department of Dermatology, University of Freiburg, Germany; *Applied Genetics Inc., Freeport; It is well established that low-dose UVB-radiation (UVBR) inhibits the APC function of murine †Division of Radiation Biology, Kanazawa University, Japan Langerhans cells in vivo. Recently, we have shown in vitro UVBR to perturb the expression of Recent studies in mice have linked UVB radiation (UVBR) induced suppression of cutaneous CD80 and CD86 on Langerhans cells, thereby inhibiting the capacity of Langerhans cells to antigen presenting cell (APC) function to UVBR produced DNA damage. The aim of this study induce allo- and antigen-specific T cell responses. DC have similar effector functions compared was to determine whether the low-dose UVBR induced suppression of the APC function of to Langerhans cells and can be easily generated in large numbers which may provide a new highly purified blood derived human dendritic cells (DC) is related to DNA damage, particularly immunologic tool for future therapy, i.e. in transplantation. CPD formation. Using a CPD specific ELISA, UVBR was shown to dose-dependently (50–200 Therefore, we asked whether low-dose UVBR of DC alters their allostimulatory capacity, and if J per m2) induce CPD in DC. Such dimer formation could be reversed by treatment of UVB- so, which are the underlying mechanisms. To address this issue, primary one-way mixed leukocyte DC with a liposome preparation containing photolyase from Anacystis nidulans (Photosomes) plus reaction (MLR) was performed using bone-marrow derived C57/BL6 DC and BALB/C photoreactivating light (PRL). By contrast, photosomes or PRL had no effect when used alone. lymphnode cells. First, we found low-dose UVB-DC to dose-dependently (50–200 J per m2) The same doses of UVBR shown to induce CPD also suppressed the capacity of DC to stimulate inhibit T cell proliferation. This effect was not due to a perturbed functional expression of the the proliferation of resting alloreactive T cells in a MLR. Importantly, Photosomes plus PRL did costimulatory molecules CD80 and CD86, since stimulation of CD28 by a stimulatory antibody not restore the allostimulatory capacity of UVB-irradiated DC. As yet an another approach to did not reverse the inhibition of T cell proliferation. In addition, cell-cycle analysis revealed no investigate the role of CPD on DC APC function we employed synthetic thymidine dinucleotides cell-cycle arrest, but an increased number of T-cells in sub-G1/0 phase, indicating apoptotic T (pTpT), previously shown to mimic some UVB induced changes in melanocytes and keratinocytes. cells. Therfore, we next examined apoptosis induction in T cells which were harvested from the However, pTpT did not affect the immune phenotype, cytokine secretion or allostimulatory MLR cocultures. We found that the T cells coincubated with UVB-irradiated DC underwent function of DC. Moreover, pTpT did not affect the proliferative capacity, cell cycle or cytokine dose-dependently apoptosis as determined by TUNEL-staining and DNA-laddering. To identify secretion of 01αCD3 ⍣ 01αCD28 activated human T cells. In summary, these experiments using the molecular mechanism of apoptosis induction, MLRs were performed with Fas-and FasL- highly purified human DC indicate that the UVBR-induced CPD formation and the suppression deficient DC and wildtype T cells. Interestingly, no diffrences were found comparing wildtype of their APC function are not directly related. DC to Fas-/FasL-deficient DC, suggesting a mechanism independent of the Fas/FasL-pathway. By using Fas-/FasL-deficient T cells an autosuicidal effect of T cells could be excluded. In conclusion, we show that low-dose UVBR of DC induces apoptosis in T cells by a mechanism independent of the Fas/FasL pathway. These results suggest that DC respond differently to UVBR compared to Langerhans cells.

525 526 Treatment of Human T Lymphocytes with 8-Methoxypsoralen Plus UVA Induces Transient But Helium-Neon Laser Treatment Induces Repigmentation in Segmental-Type Vitiligo Biologically Active Th1-Skewing Cytokine Production H. Yu, M. Wu, Y. Kao and C. Wu Y. Tokura, N. Seo, H. Yagi, H. Wakita, F. Furukawa and M. Takigawa Department of Dermatology, Kaohsiung Medical College, Kaohsiung, Taiwan; and Department Department of Dermatology, Hamamatsu University School of Medicine, Hamamatsu, Japan of Dermatology, China Medical College, Taichung, Taiwan 8-methoxypsoralen (8-MOP) plus ultraviolet A light (UVA) is suggested to shift T lymphocytes Segmental vitiligo is associated with a dysfunction of sympathetic nerves in the affected skin. from Th2 to Th1 cells. To clarify this issue, we examined the effects of 8-MOP/UVA on cytokine Segmental vitiligo is relatively resistant to treatments. Since helium-neon (He-Ne) laser irradiation mRNA expression, cytokine-secreting cell numbers, produced amounts of cytokines, and Th1/ reveals biological effects for improving nerve injury, the possibility of He-Ne laser as a potential Th2 dichotomy-relevant T cell bioactivities in peripheral blood mononuclear cells (PBMC) from application in the treatment of segmental vitiligo is worth studying. The purpose of this presentation a normal subject and a Se´zary syndrome patient. 8-MOP/UVA augmented the expression of was to study the effective-ness of He-Ne laser treatment on vitiligo and its possible mechanisms. mRNAs for interferon-01γ (IFN-01γ) and interleukin (IL)-2 2 h after treatment, whereas those Thirty patients with segmental vitiligo were enrolled in this study. Three J per cm2 was administered for IL-4 and IL-10 were decreased. The number of IFN-01γ-secreting lymphocytes was markedly for each local treatment. Patients were treated 1–2 times weekly. Marked repigmentation was increased in 8-MOP/UVA-treated PBMC 20 h after treatment, whereas that of IL-10-producing observed in 60% of patients. He-Ne laser irradiation induced nerve growth factor (NGF) cells was decreased. The amount of IFN-01γ was elevated in culture supernatants from 8-MOP- production in cultured keratinocytes and basic fibroblast growth factor (bFGF) release from phototreated PBMC and CD4ϩ T-cell clones, while IL-4 was significantly reduced in the treated cultured fibroblasts. NGF is a paracrine survival factor for melanocytes and bFGF is an effective PBMC. However, the enhanced production of IFN-01γ was found only until 3 d after 8-MOP- mitogen for melanocytes. It is reasonable to propose that He-Ne laser treatment stimulates phototreatment and its production level was rapidly declined by 5 d after the treatment. Finally, production of these factors to rescue damaged melanocytes and melanocyte proliferation in lesional 8-MOP/UVA treatment upmodulated T-cell ability to induce keratinocyte CD54 expression, a skin, therefore provoking repigmentation in segmental vitiligo. known phenomenon induced mainly by IFN-01γ. Our results show that 8-MOP/UVA has a transient but biologically active Th1-skewing action in human T cells, implying its beneficial therapeutic effect on cutaneous T-cell lymphoma as a cytokine modifier.

527 528 Analysis of Prostanoid Production by Ultraviolet (UV) Irradiated Human Blood Derived Dend- Dose–Response of Melatonin (N-Acetyl-5-Methoxytryptamine) as a Radical Scavenger on UV- ritic Cells Irradiated, IL-3 Stimulated Leucocytes M. Grewe, M. Klammer, K. Vogelsang and J. Krutmann T. Fischer, B. Kno¨ll, U. Hipler and P. Elsner Clinical and Experimental Photodermatology, Department of Dermatology, Du¨sseldorf, Germany Department of Dermatology, Friedrich-Schiller-University, Jena, Germany Prostanoids mediate the sunburn reaction and immunological changes observed after UVB Reactive oxygen species (ROS) are supposed to be involved in inflammatory UV-reactions of the irradiation (UVBR) of human skin. Epidermal keratinocytes are well known producers of skin. Clinical studies have shown that the radical scavenger melatonin exerts an UV-protective prostanoids. In contrast, for dendritic cells there is only preliminary evidence for prostanoid effect by probably quenching these radicals. This mechanism was examined in an in vitro irradiation production based on immunohistochemical detection of enzymes. We therefore assessed the model with IL-3 stimulated leucocytes and different doses of melatonin. functional capacity of DC to synthesize prostanoids. DC were generated from monocytic fractions Neutrophile granulocytes were isolated from EDTA whole blood by density gradient isolation of buffy coats by culture in presence of IL-4 and GM-CSF for 7 days. HPLC and ELISA analysis with dextran. Separated leucocytes were taken up in IL-3 stimulated buffer solution (PBS) and of supernatants of 3H-arachidonic acid (AA) prelabeled and unlabeled DC identified thromboxane the cell suspension was divided into 10 aliquots: two aliquots were treated with PBS, one left (TX)B2 and prostaglandin (PG)E2 as the major AA metabolites, while PGD2 was produced in unirradiated and one irradiated with 5 min UVB-light (280–360 nm, Max: 310 nm). The other small amounts. Lipopolysaccharide (LPS) induced secretion of both, PGE2 and TXB2. UVBR eight aliquots were treated with increasing amounts of melatonin (1, 5, 10, 20, 30, 50, 75, 100 alone was not able to significantly increase prostanoid release, while UVA1R selectively induced mmol) and irradiation was performed with UVB-light for 5 min (light source: Waldmann UV800 2 PGE2, but not TXB2 production. UVBR immediately prior to LPS stimulation resulted in UVB; distance: 30 cm; dose: 2,34–2,60 mW per cm ). Radical formation in all solutions was significant increase of TXB2 and PGE2 release as compared to LPS alone. Time kinetics for TXB2 measured in the luminometer LB 953 (Berthold, Germany) and staining with trypan blue was µ and PGE2 release paralleled each other and were maximal after 24 h. Increased prostanoid release performed to assess viability. The addition of lucigenin solution to 200 l cell suspension was was always associated with induction of mRNA for the cyclooxygenase II (CoxII): LPS treatment carried out by means of injection pumps (0,1 mmol per liter in PBS, pH ϭ 7,5, T ϭ 37°C). lead to moderate induction peaking at 4–8 h and downregulation after 24 h. Costimulation with Four-fold measurements were performed by the slow kinetics method over a 60-min period and UVBR resulted in superinduction, but not in extended expression of CoxII mRNA. In addition, calculated by means of Microsoft Excel (Astute). UVA1R lead to induction of CoxII mRNA with two maxima after 2 and 12 h. In summary, to In untreated leucocytes UV-irradiation for 5 min caused a significant higher radical formation the best of our knowledge, this is the first direct demonstration of prostanoid production by compared to unirradiated leucocytes. In irradiated leucocytes, a dose dependent suppression of human DC. UVR-induced modulation of DC prostanoid production depends on the wavelength, ROS formation by increasing dosages of melatonin was assessed in suspensions of IL-3 stimulated the type of prostanoid and on additional stimulatory conditions. Our studies indicate a previously leucocytes. The radical formation was suppressed to levels of unirradiated suspensions by 10 mmol unrecognized immunomodulatory property of DC which may be of particular relevance for T- melatonin and maximum effect was detected at a concentration of 20–30 mmol. No significant cell activation. increase of a suppressive effect was exerted with dosages of 50–100 mmol melatonin. We present an in vitro test design suitable for the evaluation of radical scavengers’ efficacy in UV- irradiated leucocytes. Melatonin was shown to be an excellent radical scavenger in a dose dependent manner. These results confirm the clinical observation of the UV-protective properties of melatonin by quenching reactive oxygen species. Further investigations will be conducted with keratinocytes. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 611

529 530 Evaluation of Apoptotic Cells Induced by Ultraviolet Light B Radiation in Epidermal Sheets Ultraviolet Irradiation Induces Beta-Galactosidase Activity Driven by Matrix Metalloproteinase-9 Stained by the TUNEL Technique Promoter in Hairless Mouse Skin H. Okamoto, K. Mizuno, T. I. K. Tanaka* and T. Horio R. Ehama Dept of Dermatology, Kansai Medical University, Moriguchi, and *Division of Cellular Genetics, Harvard University, Charlestown, Massachusetts Institute for Molecular and Cellular Biology, Osaka University, Osaka, Japan Matrix metalloproteinase-9 (MMP-9, gelatinase B) belongs to a family of highly homologous Two major components of epidermal cells, keratinocytes and Langerhans cells, are injured by Znϩϩ endopeptidases, which degrade extracellular matrix proteins such as type IV collagen and ultraviolet tight B (UVB) radiation, resulting in sunburn cell (apoptotic cell) formation, impaired elastin. In human skin, induction of MMP-9 mRNA by UVB has been reported. We studied function and a reduced number of Langerhans cells. Quantitative analysis of Langerhans cell UVR-induced endogenous MMP-9 in hairless mouse and utilized a MMP-9 reporter gene damage is usually performed using epidermal sheets, whereas that of keratinocytes has been transgenic mouse model. In hairless mouse skin, MMP-9 activity, determined by gelatin performed by counting the number of sunburn cells in vertical tissue sections. In this study we zymography, was induced 8–48 h following UVB irradiation. A hairless transgenic mouse model assessed the inﬂuences of UVB radiation on keratinocytes by apoptotic cell formation, using was produced by crossing rabbit MMP-9 promoter – lacZ transgenic mice (Mohan et al., J Biol murine epidermal sheets stained by terminal deoxy-nucleotidyt transfe rase-mediated dUTP nick Chem 273:25903, 1998) with SKH-1 hairless mice. UVB (250 mJ per cm2, 4MED) treatment of end labeling (TUNEL) technique. Ten to 75 mJ per cm2 of UVB radiation induced apoptotic the dorsal skin induced 01β-gal activity along the basement membrane. Moreover, 10–20 J per cells in abdominal skin of C3H mice. The cells were induced in 6 h after 50 mJ per cm2 of UVB cm2 UVA also induced 01β-gal activity at basal layer of epidermis 24–48 h following irradiation. irradiation with the peak in number in 24 h, 18.8 Ϯ 5.0 per mm2 and 97.7 Ϯ 17.4 per mm2, We also generated transgenic mice having the human MMP-9 promoter (–714 to ϩ19) plus lacZ respectively. One week later, the apoptotic cells were not visualized. Using confocal laser scanning gene by pronuclear injection of SKH-1 mice fertilized oocytes. Beta-gal expression in the resultant microscopy and double staining, Ia-positive cells were infrequently seen in apoptotic cells induced transgenic mice was consistent with the endogenous MMP-9 expression pattern such as in the by 50 mJ per cm2 of UVB radiation, white no apoptotic dendritic epidermal T cells were observed. bone and the wound edge of skin. Beta-gal activity was observed sporadically in individual cells Since C3H/He, BALB/C and C57/BL mice showed almost the same frequency of apoptosis in in epidermis after UVB irradiation. These results suggest that both UVA and UVB can induce epidermal sheets from 50 mJ per cm2 UVB-irradiated skin, the induction of the cells by UVB MMP-9 gene expression in mouse skin. radiation did not depend on the genetic trait of the mouse. However, xeroderma pigmentosum type A (XPA) gene-deﬁcient mice showed a greater induction of apoptotic cells (216.9 Ϯ 25.2 per mm2) by UVB radiation than XPA wild type mice (89.5 Ϯ 13.6 per mm2) and conventional mice. Compared to the assessment of apoptotic cells or sunburn cells in vertical tissue sections, the TUNEL technique with epidermal sheets appears to be a more appropriate method for quantitative evaluation of keratinocyte damage induced by UVB radiation.

531 532 Changes of RAD50 and p53 Expression by Ultraviolet B Irradiation in Skin Effects of Repeated Suberythemal Doses of UVA1, UVA2, and UVB in Human Skin S-C. Lee, W. Pyo, J. Lee, Y. Won and K. Kim M. T. Girard, R. Wehr, M. D. Lehinan, J. Wares, AJ. Stones and N. J. Lowe Department of Dermatology, Chonnam University Medical School; Department of Pharmacology, Unilever Research, Edgewater, New Jersey and Sharnbrook, UK and Clinical Research Specialists College of Dentistry, Chonnam University, Kwangju, Korea of Santa Monica, Santa Monica, California p53 is one of repair genes to remove damaged DNA, and is known to be induced by ultraviolet It has been demonstrated that chronic exposure to the ultraviolet (UV) component of sunlight B (UVB) irradiation in skin. RAD50 gene is a member of RAD52 epistasis group, and is required induces dramatic changes in human skin resulting in photodamage and with time, photoageing. for the repair of double-stranded breaks of DNA, suggesting the presence of functional relationship Although it has been reported that these changes can be induced by suberythemal doses of full- with p53 as the gene involved in cell cycle or tumor suppression. To our best knowledge, spectrum UVA and UVB, it remains unclear whether exposure to UVA1 and UVA2 has a however, no reports have been performed on RAD50 expression in the normal or ultraviolet- differential effect. To investigate this further, the buttocks of 20 subjects (Fitzpatrick skin type 11) irradiated skin. This study was aimed to evaluate changes of expression of RAD50 and p53 in the were irradiated twice weekly for 12 wk with 1/2 Minimal Erythernal Dose (MED) UVA1, UVA2, UVB irradiated skin to understand their functional relationship in skin. For time-course experiments, or UVB in a randomized, investigator blind study. Weekly clinical evaluations of subject skin 400 mJ per cm2 of UVB was irradiated to the back skin of rat, and skin sampling was performed demonstrated that UVB and UVA1 produced significantly more fine wrinkles than UVA2 at all sequentially until 48 h after irradiation. Protein and mRNA expression of the genes were evaluated time points. While UVA1 and UVB produced equal levels of erythema, UVB caused significantly by western and northern analyses. In Western blot analysis, a polyclonal antihuman meRAD50 to greater erythema than UVA2 at weeks 1–4 and 6, and UVA1 produced significantly greater RAD50 and a monoclonal DO-1 (Santa Cruz Biotech) to p53 were used as primary antibodies. erythema than UVA2 at weeks 1–5 and week 10. Changes in morphology were assessed in biopsies In northern blot analysis, mouse cDNA clone was used for mRNA expression of RAD50. obtained 1 wk after the last irradiation. Morphologic characteristics of photodamage including The results showed that RAD50 was suppressed from the early stage after irradiation, and its sunburn cell formation, inflammation, and alterations in extracellular matrix composition were expression was recovered to the normal level at 48 h postirradiation. Such change of RAD50 differentially induced. Our results demonstrate that exposure to suberythemal doses of UVA1, expression by UVB irradiation was observed in cultured cells of keratinocytes and fibroblasts. On UVA2, and UVB cause differential changes in the clinical and morphologic characteristics of skin. the contrary, p53 expression was induced from early stage after irradiation in both rat skin or cultured cells. Taken together, it is supposed that RAD50 responds differently to UVB ray in comparison with p53, and the suppression of RAD50 expression may be one of factors to cause UVB-induced skin cancers.

533 534 Dietary EPA Protects Against Mutation Induction in UVB-Exposed 01λlacZ-Transgenic Mice Use of 3-Dimensional In Vitro Human Skin Models for Photobiology Studies A. Vink, M-J. Steenwinkel, M. de Vet and L. Roza L. Roza, P. van den Berg, J. G-B. Henegouwen and A. Vink TNO Nutrition and Food Research Institute, Skin Effects Testing & Photobiology, Toxicology TNO Nutrition and Food Research Institute, Skin Effects Testing and Photobiology, Toxicology Division, Zeist, The Netherlands Division, Zeist, The Netherlands Negative health effects of exposure to (solar) ultraviolet light (UV) are manifold, the induction of Commercially available sunscreens are usually labelled with a sun protection factor (SPF) index. skin cancer being the most serious one. Whereas protective clothing and sunscreen cremes, mostly This factor is based on the endpoint erythema (sunburn). The SPF, however, does not used during the summer holidays, can provide protection, several drawbacks to this local protection indicate the level of protection against DNA damage, a relevant endpoint in view of photo- are apparent. For instance, adequate protection is only acquired if the sunscreen is applied evenly immunosuppression, -mutagenesis and -carcinogenesis. To evaluate the feasibility of 3-dimensional and frequently and in sufﬁcient amounts. Because the induction of nonmelanoma skin cancer is in vitro human skin models for photoprotection studies, we have investigated induction and repair related to the total accumulated UV dose, protection is also desirable during outdoor activities on of DNA damage UV-exposure. Human skin equivalents (Skin ZK1300 and Skinethic) as well as skin cloudy days, when sunscreens tend not to be used, to prevent further contribution to the organ cultures were used. In all models cyclobutane pyrimidine dimers were immunohistochemically accumulated UV dose. Therefore, systemic delivery of photoprotectants is a convenient and detectable after UV-exposure. The level of damage observed in the various models was highest potentially effective alternative to protect the skin against the harmful effects of UV. for Skin and lowest for human skin organ cultures. In addition, the effect of an SPF8 sunscreen In the present study we have investigated the photoprotective effect of dietary eicosapentaenoic on DNA damage induction was determined. Sunscreen application showed to protect against the acid (EPA), using the 01λlacZ-transgenic mouse model. This mouse strain contains 80 copies of induction of pyrimidine dimers, however, the level of protection was higher than expected on a transgene shuttle vector in all cells. The vector contains the bacterial lacZ-gene which encodes the basis of the SPF. Importantly, removal of DNA damage was demonstrable, indicating that the for the enzyme 01β-galactosidase. Exposure of the skin to UV can give rise to mutations in both epidermal cells in the cultures can actively repair CPD. This activity also implies that the models the host DNA and in the lacZ transgenes present in epidermal cells. The resulting altered 01β- can be used for testing of active ingredients (cosmeceuticals) in order to substantiate various claims. galactosidase is used to determine the mutation frequency. A 3-wk diet containing 15% EPA prior to UVB irradiation of the skin with 2000 J per m2 signiﬁcantly lowered mutation induction by 30%, when compared to the UV-induced mutation frequency in control mice on a 15% oleic acid diet. Moreover, DNA damage and cell proliferation have been studied in order to elucidate the photoprotective mechanism. 612 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

535 536 PUVA (8-Methoxy-Psoraten plus Ultraviolet A) Induces the Formation of 8-Hydroxy-2’- Increased Prevalence of Epidermodysplasia Verruciformis-Type HPV DNA Sequences in Hairs Deoxyguanosine and DNA Fragmentation in Calf Thymus DNA and Human Epidermoid and increased HPV 8 Antibody Levels in the Serum of PUVA-Treated Patients with Psoriasis Carcinoma Cells P. Wolf, H. Seidl, B. Binder, B. Pu¨tz, U. Schmidbauer, H. Soyer, P. Fuchs,* H. Pfister* and H. Kerl Huachen Wei, Zhisong Liu, Yuhun Lu and M. Lebwohl Department of Dermatology, Karl Franzens University, Graz, Austria; *Institute of Virology, Department of Dermatology, Mount Sinai School of Medicine, New York, New York Cologne Center for Molecular Medicine, University of Cologne, Germany The objective of this study is to investigate if 8-methoxy-psoralen (8-MOP) plus ultraviolet A Clinical follow-up studies have revealed that psoralen and UVA (PUVA)-treated patients are at (UVA) radiation (PUVA) induce oxidative DNA damage. When calf thymus DNA was incubated increased risk of developing skin cancer, however, the mechanisms of PUVA carcinogenesis are with 8-MOP and irradiated with UVA(335–400 nm), the level of 8-hydroxy-2’-deoxyguanosine (8- not completely understood at present. Human papillomavirus (HPV) has been implicated in PUVA OHdG) was substantially increased by approximately 6-fold. Formation of 8-OHdG proportionally carcinogenesis because epidermodysplasia verruciformis (EV)-type HPV DNA sequences have correlated with both UVA fluence and 8-MOP concentrations. Human epidermoid carcinoma been found in PUVA-associated tumors. To determine if PUVA treatment increases the prevalence cells were incubated with 10 µg 8-MOP per ml, followed by irradiation of 25 kJ per m2 UVA. of HPV in the skin, we investigated plucked hairs of PUVA-treated psoriasis patients with and The level of 8-OHdG increased by nearly 3-fold in PUVA-treated cells compared to 8-MOP and without a history of skin cancer as well as hairs of PUVA-untreated psoriasis patients for the UVA controls. The formation of 8-OHdG correlated with DNA fragmentation as determined by presence of EV HPV DNA sequences by a nested consensus polymerase chain reaction (PCR) spectrofluorometry. To investigate the reactive oxygen species (ROS) involved in PUVA-induced (Berkhout et al, 1995). In addition, we performed an IgG specific enzyme linked immunosorbent oxidative DNA damage, less or more specific ROS quenchers were added to DNA solution prior assay (ELISA) with L1 virus-like particles (VLPs) of the EV-specific antigen HPV8 and a HPV16 to PUVA treatment. The results showed that only sodium azide and genistein significantly ELISA to determine antibody levels in the serum of the enrolled subjects. The mean Ϯ SEM quenched PUVA-induced 8-OHdG, whereas catalase, superoxide dismutase and mannitol exhibited HPV8 antibody level (optical density value) was 0.234 Ϯ 0.052 in PUVA-treated subjects with a no effect. The quencher study with cultured cells indicated that N-acety]cysteine and genistein history of skin cancer (n ϭ 16), 0.223 Ϯ 0.058 in PUVA-treated subjects without a history of protected oxidative DNA damage as well as DNA fragmentation by PUVA treatment. Our studies skin cancer (n ϭ 12), and 0.119 Ϯ 0.023 in PUVA-untreated control psoriasis patients (n ϭ 22) suggest that PUVA treatment is able to induce the formation of 8–OHdG in purified DNA and (Student’s t-test, P ϭ 0.0326). The HPV 16 ELISA revealed that there was no statistically cultured cells and that singlet oxygen is the principle reactive oxygen species involved in oxidative significant difference in the antibody levels of this genital HPV type among the different study DNA damage by PUVA treatment. groups. The PCR studies showed that nine of 16 (56%) PUVA-treated subjects with a history of skin cancer, nine of 12 (75%) PUVA-treated subjects without a history of skin cancer, and nine of 22 (41%) PUVA-untreated control psoriasis patients were positive for HPV sequences (01χ2, n.s.). Automated sequencing disclosed the presence of a variety of HPV types, including HPV 10, 17, 22, 23, 25, RTRX7 and RTRX9. Taken together, these results suggest that PUVA increases the prevalence of EV HPV in the skin of psoriasis patients, and subsequently may lead to increased EV HPV antibody titers in the serum. These findings support the hypothesis that EV HPV may be involved in PUVA carcinogenesis; however, further studies are necessary to determine whether HPV plays indeed a pathogenic role in skin cancer formation of PUVA-treated psoriasis patients.

537 538 Microfine Zinc Oxide (Z-Cote) and Microfine Titanium Dioxide – A Critical Comparison of Successful Separation of UVB Induced Apoptosis and Necrosis in Cultured HaCaT Cells their Use as Physical Sunscreen Actives D. Gan, T. Mammone,* R. Lockshin,* K. Marenus and D. Maes D. Fairhurst, M. Mitchnick, N. Kollias, R. Gillies and S. Pinnell Estee Lauder, Melville, New York, *St. John’s University, Jamaica, New York SunSmart Inc., Wainscott, New York; Massachusetts General Hospital, Boston, Massachusetts; We have observed that UVB induces fragmentation of DNA in a biphasic manner. After an initial Duke University Medical Center, Durham, North Carolina small increase in DNA fragmentation at doses of 5–10 mJ per cm2, there is a subsequential increase Broad spectrum ultraviolet radiation protection is desirable to prevent skin cancer and photoaging. in fragmentation at higher doses of 30 mJ per cm. This first peak in DNA fragmentation correlates Physical (inorganic) sunscreens have advantages over organic sunscreens because they remain on with increased levels of other apoptotic markers. We observed that protein levels for fas, PARP, the surface of the skin, are less likely to cause irritation or allergic reactions, and are photostable. fas ligand were elevated at the same low doses of 5–10 mJ per cm2. These apoptosis markers ZnO and TiO2 are often incorrectly considered interchangeable. However, ZnO has a lower decreased during the second increase in DNA fragmentation. refractive index (1.9) in visible light compared to TiO2 (2.6) and size for size scatters light less The initial increase in DNA fragmentation and clear expression of apoptosis markers at the low efficiently (less white). Moreover, UV attentuation by microfine ZnO extends from about 260– UV dose range implies that apoptosis is occurring. The DNA fragmentation at higher UVB doses 385 nm whereas microfine TiO2 extends from about 260–360 nm. In this study microfine ZnO has reduced levels of the apoptotic protein markers and therefore may be necrosis. However, and TiO2 were formulated at varying concentrations. Particle size distribution of dispersed these higher doses of UVB increased DNA fragmentation as well as the protein involucrin, a suspensions were similar measured by X-ray disc centrifuge sedimentometry. In vivo attenuation marker of keratinocyte differentiation. was measured on human volunteers using UV reflectance spectroscopy with and without sunscreen These data suggest a clear separation of apoptosis cell death and that of terminal differentiation. (2 mg per cm2). Both pigments were evaluated at 2% and 6% wt/vol. Results mirrored in vitro It also suggests caution in the use of UVB as an inducer of apoptosis. This inducer can be UV attenuation with ZnO protection extending to about 385 nm and TiO2 extending to about nonphysiological and result in necrosis. 360 nm. Similar dispersions were measured for whitening at concentrations from 0.5% to 10% wt/vol, using a standardized ASTM test method for determining the hiding power of paints by reflectometry. Hiding power was compared at three different film thicknesses. Results showed that microfine ZnO was substantially less whitening than microfine TiO2 at all concentrations. In summary: microfine ZnO in comparison to microfine TiO2 under the same conditions (i) affords greater protection against UVA1, and (ii) demonstrates less whitening.

539 540 Loss and Induction of Transcription Factors C/EBP01β and CHOP (gadd153) in Murine Epidermis Reflectance Spectrophotometric Measurement of Human Skin Pigmentation In Vivo: an Analysis Following Exposure to UVB Light of Ethnic Variation R. Torres, S. Gonzalez and E. Maytin A. Lin and L. Meyer Dept of Dermatology, Wellman Laboratories of Photomedicine, Massachusetts General Hospital, Department of Dermatology, University of Utah School of Medicine and GRECC, SLC VA Med Harvard Medical School, Boston Center, Salt Lake City, Utah Transcription factors of the C/EBP family regulate genes involved in growth, differentiation, and Previous studies of skin pigmentation have generally used reflectometers with wavelength filters. apoptosis in the epidermis. CHOP (also called gadd153) is a member of the C/EBP-family We have used a reflectance spectrophotometer to determine reflectance at wavelengths from 385 originally identified as a UV stress-responsive gene in cultured cells. CHOP is expressed in to 750 nm with a goal of genetic analysis of pigmentation, pigment inducibility, and cancer epidermal keratinocytes in vitro and in vivo. However, UV-induced changes in CHOP expression susceptibility. Baseline and inducible pigmentation was studied in 45 people of various ethnicities, in the epidermis have not been previously characterized. Here, we examined the expression of and in 10 large nuclear families with Type I–III skin. Baseline pigmentation was assessed by CHOP and its most abundant dimerization partner, C/EBP01β, in UVB-irradiated skin. Hairless subtraction of upper inner arm data from a composite spectrum taken from vitiligo. Inducible SKH1 mice were exposed on their right flank to either 10 or 40 mJ per cm2 of UVB. At various pigmentation was assessed by subtracting spectra taken from the dorsal forearm from that of the times after exposure (immediately, 0.5 h, 4 h, 24 h, or 48 h), biopsies were taken from exposed upper inner arm. The subtracted spectra represent primarily melanin since other components such skin and from contralateral unexposed skin, fixed in formalin, paraffin-embedded, and sectioned for as blood and keratin are subtracted. These spectra showed two peaks at 450–550 nm and 580– immunostaining. Specimens were also snap-frozen for western analysis. By immunohistochemistry, 680 nm that we believe represent different melanins. Our population represented nine groups: abundant levels of CHOP and C/EBP01β were detected in unexposed skin and at 48 h post- Skin types I–IV, East Asian, Indian, Mexican, Middle Eastern, and African. Differences for the UVB. Immediately after UVB, however, significant decreases in both CHOP and C/EBP01β groups were found for baseline pigment, with areas under the curve (AUCs) ranging from 16,000 levels were observed. CHOP and C/EBP01β protein expression began to recover within 30 min, (Type I) to 74,000 (African), and ratios of the melanin peaks ranging from 0.7 (Type I) to 1.36 and by 4 h had reached or exceeded original levels, finally returning to normal by 48 h. Other (East Asians). When both AUCs and ratios are plotted, the ethnic groups clearly clustered. proteins (C/EBP01α and keratin K14) were not significantly changed. These results were Differences for inducible pigmentation were also found with ratios of 0.6 (African) to 6.4 (Type confirmed using a second technique, western blot analysis. In summary, UVB exposure causes a II) and with an inverse relationship of baseline to induced pigment. Type II was the most rapid loss of epidermal CHOP and C/EBP01β, followed by subsequent induction. Because chop heterogeneous group for both baseline and induced pigmentation, suggesting genetic heterogeneity. and the c/ebp01β genes are autoregulated at the promoter level, our results raise the possibility This is a noninvasive method to assess pigmentation differences in human skin for risk assessment that inductions of CHOP and C/EBP01β expression occur in response to rapid and selective and genetic analysis. degradation of these proteins following UVB exposure. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 613

541 542 Psoralen Adducts Targeted to Specific Sequences of the Interstitial Collagenase Gene in Human Diffuse Reflectance Spectroscopy (400–800 nm) of Skin Indicates Arterial Insufficiency Fibroblasts J. Bykowski, G. Zonios, R. Gillies, G. LaMuraglia and N. Kollias D. Oh and P. Hanawalt Wellman Laboratories of Photomedicine, Massachusetts General Hospital, Boston, Massachusetts Department of Dermatology, Stanford University School of Medicine, Stanford, California An accurate, noninvasive assessment of arterial insufficiency and the skin’s healing capacity is The ability to target photochemical adducts to specific genomic DNA sequences in cells is crucial for monitoring patients and evaluating reconstructive procedures. Diffuse reflectance important for further understanding the mechanism of DNA repair and mutagenesis in intact cells spectroscopy (400–800 nm) was used to evaluate changes in oxy- and deoxyhemoglobin during and as a potential form of gene-specific therapy. Triple helix forming DNA oligonucleotides elevation/dependency, venous outflow restriction and reactive hyperemia in limbs of 25 healthy linked to psoralen (pso-TFOs) have been designed to deliver UVA-induced psoralen adducts to volunteers. Leg dependency and venous outflow restriction of the arm caused equivalent, increased two distinct sequences within the collagenase gene. A ligation PCR assay demonstrates that deoxyhemoglobin concentrations; the higher total hemoglobin concentration evidenced by color adducts form in both purified genomic DNA and intact cells. The single adducts formed by pso- change in the skin. Upon relief from tourniquet ischemia, a rapid inflow of oxyhemoglobin was TFOs differed from the multiple adducts produced by free psoralen reacting with numerous other observed, achieving 50% of the maximum oxyhemoglobin concentration within 6 Ϯ 5 s in the sites within the gene, and pso-TFOs to one target did not interact with the other, indicating a leg and 10 Ϯ 9 s in the arm. Oxyhemoglobin levels returned to baseline levels within 3 min in high level of specificity. Intact fibroblasts were efficiently transfected with mixtures of cationic the arm, and near-baseline levels within 4 min in the leg. These findings correlate with the lipids and pso-TFOs at concentrations well below their cell-free dissociation constants. Five previously published changes in blood gas levels and near-infrared assessments of hemoglobin and minutes following UVA, adducts were readily observable in repair deficient fibroblasts from myoglobin under similar constraints. This demonstrates that changes in the arterial supply are xeroderma pigmentosa complementation group A but not in normal human or HT1080 fibroblasts, intimately reflected in the skin microcirculation. Compared to this ‘‘normal’’ signature response, suggesting that such adducts are rapidly removed in repair-proficient cells. Therefore, pso-TFOs the perfusion of diseased or compromised tissue can be assessed noninvasively, using the skin as a are promising reagents for investigating the repair of strategically placed psoralen photoadducts in reporting organ. intact cells. Moreover, their utility in gene therapy will most likely depend upon site-specific mutagenic processing rather than sustained inhibition of DNA metabolism.

543 544 Nicotine Reduces Keratinocyte Migration Distance by Altering Intracellular Calcium Levels via Use of Alpha Hydroxy Acids Increases Oxygen Utilization by Skin Receptor-Mediated Pathway C. Penksa, U. Santhanam and R. Weinkauf S. Zia, A. Ndoye and S. Grando Unilever Research, Edgewater, New Jersey Department of Dermatology, University of California, Davis, California Alpha Hydroxy Acids, particularly, lactic and glycolic acids, have been shown to be effective in Poor re-epithelialization of skin wounds in nicotine (Nic) users is conventionally attributed to improving the appearance of photoaged skin. It is not clear however, how these benefits are Nic-induced alterations in cutaneous blood supply. However, the outgrowth of human keratinocytes mediated. AHAs such as lactic acid are integral parts of biochemical pathways in the cell and it is (KC) in vitro does not require serum, suggesting direct effects of Nic which can be mediated by conceivable that they affect cellular metabolism. The effect of AHAs on oxygen utilization by nicotinic acetylcholine receptors (nAChR) expressed on the cell membrane of KC. In this study, human skin in vivo was examined using transcutaneous oxygen monitors. Treatment of skin with we used agarose gel keratinocyte outgrowth system to measure the migration distances of the KC 8% lactic acid cream for 1.5 h resulted in a decrease in transcutaneous oxygen relative to vehicle that were exposed for 10 d to different concentrations of Nic given alone or in combination with cream, indicating that more oxygen was utilized by skin treated with lactic acid. Similar results its antagonist mecamylamine (Mec). Nic decreased outgrowth of KC in a dose-dependent fashion. were seen upon long-term treatment with 8% glycolic acid cream for 12 wk. The results of both While the 100 µM dose of Nic decreased outgrowth to 71% Ϯ 12% (P Ͻ 0.05), it produced no studies suggest that AHAs may function, in part, by stimulating aerobic metabolism to ultimately significant effect on keratinocyte numbers and viability, suggesting that mechanisms other than generate more energy to support cellular processes. toxicity were involved. Since Nic-induced reduction of keratinocyte outgrowth could be abolished by Mec, the effects of Nic were mediated by activation of keratinocyte nAChRs. The receptor- mediated nicotinic effects on KC involve changes in the transmembrane influx and intracellular metabolism of calcium. Therefore, we studied the effects of nicotinic drugs on the concentrations ϩϩ of intracellular free calcium ([Ca ]i) using ratiometric image analysis with the calcium sensitive dye fura-2AM. Activation of keratinocyte nAChR with Nic or epibatidine elicited an immediate ϩϩ ϩϩ transient rise of [Ca ]i followed by its gradual increase. The Ca -permeable ACh-gated ion channels were visualized on the cell surfaces of KC both in vitro and in vivo in indirect immunofluorescence experiments using antibodies to the 01α3, and 01α7 nAChR subunits. We propose that these subunits form the ACh-gated ion channels that mediate the deleterious effects ϩϩ of Nic on keratinocyte migration and changes in the [Ca ]i levels. Thus, a receptor-mediated ϩϩ increase in [Ca ]i may provide a biochemical mechanism for Nic-induced reduction of keratinocyte migration in culture, and delay re-epithelialization of cutaneous wounds.

545 546 The Effects of Retinol Mixture on UVR Irradiated Hairless Mouse Skin The Distribution of Hair Density and Hair Diameter in Normal Women – Implications for Female J. Hwang, Y. Cho, S. Lee, W. Park, J. Cho, J. Oh, M. Kim, J. Lee and Y. Sim Pattern Hair Loss Skin and Applied Research Institute, Pacific R&D Center, Kyounggi-do, Korea P. Birch and A. Messenger Skin photoaging is a prime example of damage to the skin by the sunlight consisting of UVB and Department of Dermatology, Royal Hallamshire Hospital, Sheffield, UK UVA. Skin with already progressed photoaging could be improved clinically and histologically by The frequency of clinically apparent female pattern hair loss is age-dependent, increasing from topical application of retinoids. Unique action mechanism of retinoids which has not been fully about 10% in women under the age of 50%–40% in women aged over 60. The aim of this study understood includes neosynthesis of collagen, enhancement of mitogenesis of keratinocytes, was to quantify and characterize hair diameter in the normal female population across a wide age reduction of matrix metalloproteinases synthesis and generation of new blood vessels. In order to range. We studied 350 women, aged 16–99, attending a dermatology clinic with complaints explore any compounds that have synergistic effects with retinol for their in vivo efficacy, we unrelated to hair loss. Scalp hair density was measured from macrophotographs of a 0.75-cm2 treated several proprietary compounds with retinol onto the hairless mouse, which had been UV- circle on the frontal scalp. In each subject the major axis diameter was measured in 20 randomly irradiated for 8 wk, with UV only, UV ϩ vehicle, UV ϩ retinol-applied mice for their references. selected hairs from the same site. After 5-wk treatment, the treated mice skins were analyzed for their histological changes by H&E Mean hair density remained stable in the sample population up to age 50 (323 hairs per cm2 SD staining, type I procollagen synthesis, type IV collagen and MMP-1 synthesis between the different 56) and thereafter declined (mean 214 hairs per cm2 SD 73 in women over 69 y). Hair density groups of mice. One compounds showed remarkable improvement in the histology, superior to in women under 50 was distributed in a Gaussian fashion. The majority (73%) of women classified retinol-treated group, in terms of keratinocyte proliferation/differentiation, collagen synthesis and clinically as having hair loss had hair density within 2 SD of the mean. Macrophotographs of reduction of MMP-1 level. women with high hair density showed multiple hairs (3–5) emerging from single or closely grouped follicular orifices. In low hair density, individual follicles contained only one or two hair shafts. Within the sample population as a whole, hair diameter showed a bimodal distribution with two major peaks at 60 µm and 80 µm. Low hair density was associated with a greater reduction in large diameter hairs than in small diameter hairs. In the lowest quartile for hair density the numbers of large diameter and small diameter hairs were 42% and 75%, respectively, of the numbers present in the highest quartile. These results confirm that scalp hair density in women declines with increasing age, particularly after the age of 50. The reduction in hair density appears preferentially to affect large diameter hairs within follicular units. Most women with female pattern hair loss have scalp hair density which falls within the ‘‘normal’’ range. The Gaussian distribution of hair density in women strongly suggests that it is a multifactorial trait. 614 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

547 548 Pyranine as a Marker for Hyperkeratotic Lesions Aged Keratinocytes Fail to Undergo Apoptosis Following Ultraviolet B Irradiation A. Pagnoni, D. Kligman, T. Stoudemayer and A. Kligman D. Began, S. Hurwitz and D. Spandau SKIN Incorporated, Conshohocken, Pennsylvania Departments of Dermatology and Biochemistry & Molecular Biology, Indiana University School The aim of this study was to detect subclinical lesions of abnormal cornification. We applied to of Medicine, Indianapolis, Indiana the face of photodamaged subjects a yellow fluorescent ink containing pyranine, a water-soluble In the epidermis, keratinocytes are chronically exposed to a powerful DNA-damaging agent, fluorescent pyrene dye, for 20 min followed by washing. Digital fluorescent photos were taken ultraviolet light (specifically UVB). The appearance of cutaneous malignancies dramatically increases immediately after removal of the dye. Two-mm punch biopsies were taken from spots that in elderly individuals, and it has been previously reported that the capacity to repair damaged retained the fluorescent dye. DNA declines with age. We proposed that the increase of epidermal tumors in the elderly may Fluorescent photos showed a well-defined retention of the pyranine ink in hyperkeratotic sites. be due to the inability of aged epidermis to repair or rid itself of genetically damaged keratinocytes; The spectrum of hyperkeratotic lesions ranged from subclinical to clinically visible seborrheic and therefore, aged keratinocytes may also be deficient in their ability to undergo apoptosis following actinic keratoses. high doses of UVB radiation. We isolated keratinocytes from the sun-protected epidermis of This fluorescent marker has clinical value in revealing distribution of early hyperkeratotic lesions young adult (20–28-y-old) and geriatric (greater than 65-y-old) individuals. These keratinocytes of the photoaged face. were grown in vitro and irradiated with various doses of UVB. Following UVB irradiation, the induction of DNA repair and apoptosis was monitored by TUNEL, internucleosomal DNA ladder, FACS, and annexin V analyses. The induction of DNA repair by low doses of UVB was significantly impaired in geriatric keratinocytes. In addition, there was no induction of apoptosis by high doses of UVB in geriatric keratinocytes. We also grew keratinocyte cultures isolated from neonatal foreskins in vitro until they reached senescence. We then compared the response of the senescent keratinocytes and early passage keratinocytes to UVB irradiation. As seen in the intrinsically aged keratinocytes, senescent keratinocytes failed to undergo apoptosis following UVB irradiation. We conclude that the inability of geriatric keratinocytes to respond appropriately to UVB irradiation contributes to their increased malignant potential.

549 550 An Autoimmune Skin Disease With Presence of Simultaneous Acantholysis Between Keratinocytes The Distribution of Pemphigus Vulgaris-IgG Subclasses and their Reactivity to Desmoglein 1 & and Separation at the Basal Membrane Zone of the Skin 3 in Pemphigus Patients and their First Degree Relatives A-V. Ana Maria, L. Wlater and H. Ken* D. Kricheli, M. David, M. Frusic-Zlotkin,* D. Goldshmit,* M. Rabinov, J. Sulkes,† B. Michel‡ Section of Dermatology, Hospital Universitario San Vicente de Paul, University of Antioquia and Y. Milner* (U de A.), Medellin, Colombia and *Department of Dermatology and Syphilology, Wayne Dermatology Department, Rabin Medical Center – Belinson Hospital, Tel. Aviv University, Israel; State University *Myers Skin Biochemistry Laboratory, Hebrew University of Jerusalem, Israel; †Epidemiology The hallmark of the autoimmune bullous skin disease is the presence of autoantibodies that directly Unit, Rabin Medical Center – Belinson Hospital, Petah -Tiqwa, Israel; ‡Cutaneous Pathology or indirectly are involved with the acantholysis between keratinocytes (as in the case of pemphigus) and Immunofluorescence Laboratory, AMRIPATH, Beachwood, Ohio or at the basal membrane zone (as in bullous pemphigoid). Currently, no descriptions of an Pemphigus vulgaris autoantibodies (PV-IgG) were found in sera of first degree relatives of autoimmune skin disease with presence of a simultaneous epidermal acantholysis and basal pemphigus patients at a rate of 40%–70%. The aim of this study was to determine whether a membrane zone separation have been reported. After a six year case-control study in 50 people different distribution and reactivity of PV-IgG subclasses in PV patients, compared to their first affected by an endemic pemphigus foliaceus-like disease from one focus in El Bagre (a small degree relatives, may account for the rare occurrence of familial pemphigus. The study group tropical mining village of Colombia), we demonstrated that more than 50% of patients showed included 25 PV patients, 55 unaffected family members and 56 sera of healthy individuals. Indirect clinical features of Senear–Usher syndrome. In these, we found, the presence of immunostain immunofluorescence (IIF) staining and western immunoblotting (WB) technique were used to between keratinocytes and also at the basal membrane zone by immunofluorescence, and the determine total PV-IgG, PV-IgG subclasses and their reactivity to desmoglein (Dsg) 1 & 3. By presence of liquefaction of the basal membrane zone in 30% of them by histopathology. In order IIF staining, circulating PV-IgG were found in 64% of the patients, 15% of the relatives and none to corroborate these light microscopic findings with electron microscopy, this study was carried out. of the control; by WB the results were 91%, 49% and 15%, respectively. PV-IgG subclasses 1–3 Samples from people affected by endemic pemphigus foliaceus-like disease from El Bagre were were found in similar distribution among patients and their relatives. On the other hand, PV-IgG studied by electron microscopy. 4 was found in 62% of the patients (and 71% in active diseased patients), in one relative and was We detected in people affected by endemic pemphigus foliaceus from El Bagre the presence of absent in healthy individuals. PV-IgG 1 & 2 were found to react mainly with Dsg3, PV-IgG 3 acantholysis between keratinocytes and also separation at the lamina lucida of the hemidesmosomes. showed in addition more reactivity with Dsg1, while IgG 4 reacted almost exclusively with Dsg3 Presence of two classes of intracellular vesicles were also detected. One filled with an electrondense and Dsg1. The noncomplement fixing IgG 4 plus at least one complement fixing PV-IgG subclass material which resembled an immune complex and deposited near the desmosomes. Another appear to be involved in the pathogenesis of the disease. The absence of PV-IgG 4 among PV- substance, whose nature remains unknown, was also detected. IgG carriers relatives may be linked to the lack of the active disease in this group. The role of This is the first report of an autoimmune skin disease with presence of simultaneous acantholysis PV-IgG4 in pathogenesis is under study. between keratinocytes and the separation through the lamina lucida.

551 552 IL-101α and TNF-01α are the Main Cytokines Involved in Acantholysis IL-10 Immunomodulation in Treatment of Pemphigus C. Feliciani, P. Toto, P. Amerio,* S. M. Pour, G. Coscione, G. Shivji,† B. Wang† and D. N. Sauder† P. Toto, C. Feliciani, P. Amerio, H. Suzuki,* G. Shivji,* B. Wang,* V. B. Altun,* D. Woodley† Department of Dermatology, University ‘‘G.D’Annunzio’’, Chieti, Italy; *Department of Dermato- and D. Sauder* logy, University of Ancona, Italy; and †Sunnybrook HSC, University of Toronto, Canada Department of Dermatology, University ‘‘G.d’Annunzio’’, Chieti Italy, Department of Dermatology Several studies have suggested that cytokines are involved in the pathogenesis of PV. To further †North-western University, Chicago, Illinois and *Department of Dermatology, University of clarify the role we examined cytokines in 19 patients demonstrating characteristic clinical, Toronto, Canada pathologic and immunopatological finding of PV. In situ immunolabelling for interleukin-101α Pemphigus Vulgaris (PV) is an autoimmune bullous skin disease characterized by antibodies (IL-101α) and tumor necrosis factor-a (TNF-01α), demonstrated the presence of the two cytokines directed against the desmosomal cadherin desmoglein-3. Treatment to date has relied on broad in lesional and perilesional areas. Results were confirmed by RT-PCR demonstrating overexpression spectrum immunosuppressive or anti-inflammatory therapy. Specific immunotherapy is hampered of both cytokines in vivo. To study the role of these cytokines in the pathogenesis of PV an in vitro by a lack of understanding as to precise pathogenesis. We have gained further incite into the and in vivo study was performed. The results of in vitro acantholysis using normal keratinocytes pathogenesis by utilizing passive transfer experiments with PV IgG in gene targeted mutant mice. treated with PV-IgG demonstrated increased IL-01α and TNF-01α. The potential pathogenic Our results demonstrated that IL10–/–mice are significantly more sensitive to the development role of these mediators was demonstrated by an in vitro blocking study. A combination of anti-IL- of PV lesions than wild type mice (42% vs 10%). Subsequently we demonstrated that recombinant 01α and anti-TNF-01α antibodies inhibited in vitro acantholysis. To further evaluate the role of IL-10 was able to suppress the development of the disease in normal and IL-10–/– mice. To IL-1 and TNF in pemphigus we utilised passive transfer studies using IL-1 (ICE–/–, IL-101β–/– further prove a role of this anti-inflammatory cytokine in the process of acantholysis we performed ) and TNF-01α receptors mutant mice (TNFR1R2). IL-1 and TNF mutant mice demonstrated a passive transfer of PV IgG in IL-10 transgenic mice. Supporting the result in IL-10–/–, that IL- a decreased susceptibility to pemphigus. Our data support the role of cytokines IL-1 and TNF- 10 plays a protective role in PV, we were able to show that IL-10 transgenes were relatively 01α in the pathogenesis of PV. resistant. Taken together, these data suggest a protective role of IL-10 in PV and support a potential therapeutic role for IL-10 in treatment of pemphigus. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 615

553 554 Epidermolysis Bullosa with Muscular Dystrophy: A Novel Premature Termination Codon and De Analysis of the Humoral and Cellular Immune Reactions of Patients with Bullous Pemphigoid Novo Deletion Mutations in the Plectin Gene M-S. Lin, G. J. Giudice and L. A. Diaz F. Rouan, L. Pulkkinen, S. LaForgia, G. Meneguzzi, P. Hyde, D-U. Kim and J. Uitto Departments of Dermatology and Biochemistry, Medical College of Wisconsin, and the VA Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Philadelphia, Medical Center, Milwaukee, Wisconsin Pennsylvania; Faculte´ de Me´dicine, INSERM, Nice, France; Department of Pathology, St. Bullous pemphigoid (BP) is a cutaneous autoimmune disorder characterized by subepidermal Barnabas Medical Center, Livingston, New Jersey blistering and autoantibodies against two hemidesmosomal proteins, BP180 and BP230. We Epidermolysis bullosa with late-onset muscular dystrophy (EB-MD) is a hemidesmosomal variant previously showed that the NC16A domain of BP180 harbors the major BP-associated extracellular of EB due to mutations in the plectin gene. Immunofluorescence of the patient’s skin and muscle epitopes, and antibodies to the corresponding region of murine BP180 trigger a BP-like skin biopsies is usually negative for staining with antibodies recognizing plectin, a large cytoskeleton disease in a passive transfer mouse model. In this study we further characterized the humoral and associated anchorage protein. In this study we report the molecular basis of EB-MD in three cellular immune response in 12 BP patients by assessing the autoantibody and T cell reactive sites families. In two families, the probands were newborns with neonatal blistering with no evidence on BP180 using immunoblotting and cell proliferation assays. We demonstrated that T cells and for muscle weakness, while the third one was an adult with skin and muscle involvement. autoantibodies from nine of the 12 BP patients reacted with fusion proteins (FP) encompassing Peripheral blood DNA was isolated and examined by heteroduplex scanning strategy, protein the NC16A region. The three BP patients who were unreactive with the BP180 FP reacted with truncation test (PTT), and/or direct sequencing of the plectin gene (PLEC1). One of the probands BP230, but not BP180, by immunoblotting with an epidermal extract. Both autoantibodies and was homozygous for a R2465X nonsense mutation, while another one was compound heterozygote T cells from the nine BP180-reactive BP patients specifically recognized the same subfragments E2005X/K4460X. The K4460X mutation was not present in either one of the parents, thus being of the NC16A fusion proteins (AA#507–520 or AA#521–534), suggesting that both T and B a de novo finding. The third family was a compound heterozygote for two deletion mutations cell epitopes are located in the same region of the BP180 antigen. Additionally, we have generated 5083delG/2756del21, the latter mutation extending from –9 to ϩ12 at the intron 22/exon 23 T cell lines and T cell clones (n ϭ 12) from three of these patients that specifically recognize the border, thus being in-frame and resulting in deletion of four amino acids. Also, the 5083delG NC16A peptide. By RT-PCR, the activated BP T cell lines and clones were shown to express mutation was not present in the parents. The stop codon mutations are predicted to result in the IL-2, IL-4, IL-5, IL-6 and IFN-01γ. These findings suggest that BP180-activated T lymphocytes synthesis of a truncated protein lacking the carboxy-terminal globular domain of the protein, from BP patients secrete a mixed Th1/Th2 cytokine profile. This information will prove valuable while the in-frame deletion presumably affects a critical domain of the plectin molecule. Plectin in dissecting the autoimmune responses underlying the development of BP. deficiency is associated with muscular dystrophy, the onset of muscle involvement varying from 2 to 35 y of age. Thus, molecular diagnostics of the plectin gene provides prognostic value in evaluation of these patients who appear to be at risk for development of muscular dystrophy.

555 556 The Paraneoplastic Pemphigus Antigens, Envoplakin and Periplakin, are also Recognized by Structural Defects of the BP180 Protein Caused by a Point Mutation (G627V) that is Linked to Pemphigus Vulgaris and Pemphigus Foliaceus Autoantibodies a Novel Form of Junctional Epidermolysis Bullosa S. Kazerounian, S. Aho, K. Rothenberger, H. Nousari, G. Anhalt, M. Mahoney and J. Uitto M. Olague-Marchan, M. K. Hacker, L. A. Diaz, S. S. Twining and G. J. Giudice Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Philadelphia, Departments of Dermatology and Biochemistry, Medical College of Wisconsin, and VA Medical Pennsylvania, and Department of Dermatology, Johns Hopkins University, Baltimore, Maryland Center, Milwaukee, Wisconsin Pemphigus vulgaris (PV), pemphigus foliaceus (PF) and paraneoplastic pemphigus (PNP) are BP180, or collagen XVII, was first identified as a major target of autoantibodies produced by autoimmune skin blistering diseases, and the pathogenic autoantibodies in PF and PV recognize bullous pemphigoid patients. When passively transferred into neonatal mice, antibodies to this the desmosomal adhesion molecules, desmogleins (Dsg) 1 and 3, respectively. In PNP, the protein trigger an inflammatory subepidermal blistering phenotype that closely mimics bullous autoantibodies recognize proteins belonging to the plakin family, including envoplakin, periplakin, pemphigoid. Structural studies indicate that BP180 is a transmembrane homotrimeric protein with bullous pemphigoid antigen 1, plectin and desmoplakin. It was recently shown that PNP sera also a C-terminal ectodomain that forms a series of collagen-like triple helices. As is typical of contain pathogenic antibodies against Dsg3 and that PF antibodies recognize a 185–195 kDa collagenous proteins, the BP180 ectodomain is relatively resistant to proteolysis. McGrath and keratinocyte protein. In this study, we demonstrate that PF and PV patients’ sera recognize coworkers have identified a single G to V substitution (G627V) located within collagen domain envoplakin and periplakin. We utilized bacterially expressed GST-plakin linker fusion proteins in 15 that is associated with a novel form of junctional epidermolysis bullosa. This disease is Western blotting and ELISA. Several PNP, PF and PV sera recognized envoplakin and periplakin characterized by skin blisters, partial alopecia and loss of dentition. Based on immunofluorescence linker domains in Western analysis, and this result was confirmed by ELISA assays. The data data, the G627V mutation appears to lead to the loss of a segment of the BP180 ectodomain but shown here demonstrate that some PNP as well as PV and PF sera recognize common antigens, the retention of the endodomain in basal keratinocytes. To facilitate the structural analysis of this and the involvement of these antibodies may modify the severity of the disease progress. mutant form of BP180, we have engineered the G627V mutation into a construct that allows for the expression of the BP180 ectodomain as a secreted protein in the human 293 kidney cell line. We have shown by HPLC and SDS-PAGE that both the normal and mutant forms of BP180 form collagen-like triple helices with similar melting temperatures. Analysis of tryptic digestion products reveals that, compared with the wild type form, the mutant BP180 protein contains an additional highly sensitive proteolytic site that maps within the region of the mutation. These findings support our hypothesis that this Gly-to-Val substitution causes a local destabilization of the triple-helix, which, in turn, exposes sensitive residues to the effects of trypsin in vitro and possibly to those of extracellular proteases in vivo.

557 558 A Large in Frame Deletion in the Cytoplasmatic Domain of BP180 in a Patient with an Detailed Mapping of BP180 Epitopes Recognized by Herpes Gestationis IgG Epidermolysis Bullosa Simplex Phenotype M-S. Lin, C-L. Fu, M. Gharia, M. Marchan, M. Hacker, K. Harman, B. Bhogal, M. Black, L. M. Huber, L. Borradori, E. Rugg, E. Lane, L. Bruckner-Tuderman and D. Hohl Diaz and G. Giudice Departments of Dermatology, Lausanne, Geneva, Switzerland; Mu¨nster, Germany; and Department Departments of Dermatology and Biochemistry, Medical College of Wisconsin, and the VA of Anatomy and Physiology, Dundee, UK Medical Center, Milwaukee, Wisconsin, and St.John’s Institute of Dermatology, St. Thomas’ BP180 or collagen XVII is a structural hemidesmosome molecule with a globular cytoplasmic Hospital, London, UK domain and a large extracellular collagenous domain. We report a male patient born to Herpes gestationis (HG) is a subepidermal bullous dermatosis associated with pregnancy that is nonconsanguineous parents with bullous lesions on the trunk, face and hand consistent with a characterized by autoantibodies against the hemidesmosomal glycoprotein BP180. Previously, we generalized variant of epidermolysis bullosa simplex. Histology revealed dermo–epidermal cleavage. have shown that autoantibodies and autoimmune T lymphocytes from two HG patients recognize Electron microscopy showed normal hemidesmosomes, but fine and reduced keratin filaments the MCW-1 antigenic region (AA #507–520), which is located within the NC16A domain of with a missing attachment at the hemidesmosomes. Antigen mapping on frozen sections the BP180 ectodomain. Here, we analyzed the sera of a large group of HG patients (n ϭ 40) by demonstrated keratins on the top and laminin 5 (GB3) on the floor of the blister and normal immunoblotting to further define the sites on BP180 that are targeted by HG autoantibodies. expression of plectin, integrins β4 and a6, small heparan sulphate proteoglycan, and collagen VII. Similar to our recent findings with BP sera, a high percentage of HG sera (93%) were found to Sequencing of K5, 14, and 15 cDNA from this patient did not provide any evidence for a possess IgG reactivity with sec180e, a 120-kDa recombinant protein encompassing the entire causative keratin mutation. However, staining with antibody 1 A8C directed against the intracellular extracellular domain of BP180. Sera from patients with unrelated skin disorders and normal domain of BP180 was absent but normal with antibodies 233 and 2D2 recognizing epitopes in controls were negative in this assay, supporting the conclusion that immunoreactivity to sec180e the extracelluar BP180 domain. Accordingly, northern blot analysis of cultured keratinocytes with is a specific marker of the subepidermal autoimmune bullous disorders. After depletion of a3ЈBP180 probe indicated that this patient does not express any normal BP180 mRNA but immunoreactivity against NC16 A, the HG sera no longer reacted with sec180e, indicating that contains only mRNA with a lower molecular weight. Moreover, using a 5Ј probe revealed that the major HG-associated epitopes on BP180 are restricted to the NC16A domain. The vast the smaller size mRNA is due to a large deletion at the 5Ј end. This was confirmed by RT-PCR majority of the HG sera [34 of the 37 BP180-positive sera (92%)] reacted with the MCW-1 cloning of the cDNA showing a 1171-bp deletion from nucleotides 158–1329 (Genbank M91669) stretch. Eleven HG sera (including the three that were sec180e-positive but MCW-1-negative) which corresponds exactly to the exclusion of exons 3 through 15 from the BP180 cDNA. This recognized one or both of two antigenic sites located just downstream of MCW-1. In summary, deletion restores the reading frame and yields a shortened protein missing the intracellular region we have identified three major extracellular epitopes on BP180 that are recognized by HG sera. from isoleucine 18 to asparagine 407. This is consistent with our data with the 1 A8C antibody These findings support the hypothesis that an autoimmune response to the BP180 NC16A domain of which the epitope has been mapped to amino acids 113–201. These findings suggest that the is a crucial step in the pathogenesis of HG. intracellular domain of BP180 might be functionally very important for the molecular interaction with keratin filaments in the basal layer in vivo 616 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

559 560 IgA Anti-Transglutaminase Antibodies in Patients with Dermatitis Herpetiformis (DH) Type VII Collagen Gene (COL7A1) Mutations in Nine Families with Dystrophic Epidermolysis R. Hall, F. Bao,* R. Streilein, G. Eisenbarth* and A. Smith Bullosa (DEB) Durham VA and Duke University Medical Centers, Durham, North Carolina; and *University A. Kon, L. Pulkkinen, A. Ishida-Yamamoto, I. Hashimoto and J. Uitto of Colorado Health Sciences Center, Denver, Colorado Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Philadelphia, DH is characterized by intensely pruritic blistering skin lesions, cutaneous granular IgA deposits, Pennsylvania; Department of Dermatology, Hirosaki University School of Medicine, Japan; and an associated, mostly asymptomatic, gluten sensitive enteropathy (GSE). The lack of gut (GI) Department of Dermatology, Asahikawa Medical College, Japan symptoms in DH patients contrasts with isolated GSE (celiac disease), even though the small DEB is an inherited mechano-bullous disorder characterized by fragility of the skin and mucous bowel lesion in both patient groups is very similar morphologically. Up to 100% of isolated GSE membrane. Mutations in COL7A1 have been shown to underlie different variants of DEB. To patients have antiendomysial antibodies, and the autoantigen is very likely tissue transglutaminase facilitate further refinement of genotype/phenotype correlations in DEB, we have examined the (tTG). Given the clinical disparity between the GI disease in DH and isolated GSE patients, we molecular basis of DEB in nine families, seven recessively (five Hallopeau-Siemens type and two sought to determine whether tTG IgA autoantibodies (ABs) were present in patients with DH as mitis type) and two dominantly (Cockayne-Touraine type and pretibial type) inherited. Mutation they have been reported in isolated GSE subjects. Sera from 27 DH patients and 18 patients with detection strategies consisted of PCR amplification of COL7A1 from genomic DNA, followed either linear IgA or linear IgG skin deposits were tested for tTG IgA ABs using a radioimmunoassay, by heteroduplex analysis and direct nucleotide sequencing. The results revealed 16 allelic mutations, utilizing in vitro transcribed and translated human transglutaminase. Gut lavage-derived secretions 11 of them being novel, previously unpublished. Thus these variants of DEB are allelic, and subtle from nine DH patients and five controls were tested also. Eleven DH patients underwent small differences in the clinical presentation may reflect the precise positions of the mutations along the bowel mucosal biopsy (SBBx). Twenty of 27 (62%) DH patients tested positive for serum tTG type VII collagen molecule. The genetic information was also used for prenatal testing in a family IgA ABs. Three DH patients with negative assays had SBBx performed, revealing either partial at risk for recurrence of a Hallopeau-Siemens type of recessive DEB. These data contribute to or subtotal villous atrophy. Two patients with linear IgA deposits had serum tTG IgA ABs; the the expanding database of COL7A1 mutations in DEB. remaining 16 of 18 were negative. tTG IgA ABs were found in the gut lavage of four of nine DH patients, whereas all control subjects were negative. Serum and gut tTG IgA Abs were concordant in five of six DH patients. tTG IgA ABs are present in only 62% of DH patients despite the universal presence of GSE. All three patients without serum tTG IgA ABs that underwent SBBx had villous atrophy. We did observe a strong correlation (83%) between the antibody results in serum and gut secretions of those DH patients who were tested for both. These results suggest that tTG IgA ABs are not sufficiently sensitive to be diagnostic for the associated GSE in DH patients, despite utilization of a highly sensitivity radioassay.

561 562 Identification of Laminin-01β3 Chain as a New Target Antigen in Auto-Immune Blistering The Role of Neutrophil Elastase, Gelatinase B, and Plasmin/Plasminogen Activators in Pemphigus Skin Diseases Foliaceus and Pemphigus Vulgaris in Mice R. Ghohestani, J. Nicolas, D. Aberdam, A. Claudy and J. Uitto Z. Liu, X. Zhou, X. Ding, R. Chen, S. Shapiro, R. Senior, G. Giudice, J. Fairley and L. Diaz Immunodermatology Unit, Department of Dermatology and Cutaneous Biology, Jefferson Medical Department of Dermatology, Medical College of Wisconsin; VA Medical Center, Milwaukee, College, Philadelphia, Pennsylvania; INSERM U385, Nice; INSERM U999 and Department of Wisconsin; and Department of Internal Medicine, Washington University, St. Louis, Missouri Dermatology, Claude Bernard University, Lyon, France Pemphigus foliaceus (PF) and pemphigus vulgaris (PV) are two autoimmune blistering diseases. Laminin-5 is composed of three distinct gene products; the 01α3, 01β3 and 01γ2 chains. A subset Acantholysis (cell–cell detachment) in PF is restricted to the subcorneal layer of the epidermis, of patients with cicatricial pemphigoid (CP) has previously been shown to have antibodies directed whereas both skin and mucosal epithelia are targeted in PV, and acantholysis is observed at the against the 01α3 chain. Here, we present two CP cases with IgG auto-antibodies directed against suprabasilar areas of these epithelia. The autoantigens for PF and PV are the 160-kDa desmoglein- the laminin-5, specifically the 01β3 chain. The in vivo bound IgG antibodies from lesional skin 1 (Dsg1) and 130-kDa desmoglein-3 (Dsg3), respectively. Anti-Dsg1 and anti-Dsg3 autoantibodies samples were localized by double immunofluorescence microscopy along the dermal-epidermal have been shown to be pathogenic in passive transfer mouse models. It has been suggested that junction between the extracellular part of BP180 (NC16) and type VII collagen. The patients’ the acantholysis in PV and PF is mediated by the activation of certain proteases. In this study we circulating antibodies bound the dermal part of salt-split skin in a pattern similar to anti01β3 investigated the role of neutrophil elastase (NE), gelatinase B (GelB), and the plasmin/plasminogen antibody. The patients’ IgG antibodies exclusively bound a unique 140 kDa band comigrating activator (PA) system in PF and PV using in vivo IgG passive transfer experiments. The extracellular with the 01β3, as identified by a specific antibody. There was no antibody reactivity against the domains of Dsg1 and Dsg3 were expressed in the baculovirus system and affinity-purified using a 01α3or01γ2 chains using purified laminin-5 as a substrate. Furthermore, the patients’ auto- Ni-NTA agarose column. Soluble recombinant domains of Dsg1 and Dsg3 were then used to antibodies did not recognize the 180 or 230 kDa bullous pemphigoid antigens or type VII collagen affinity-purify anti-Dsg1 and anti-Dsg3 autoantibodies from PF and PV sera. Mice containing by western blot assay. The anti-01β3 reactivity of the patients’ sera was further confirmed using targeted disruptions of the NE, GelB, tPA (tissue-type), or uPA (urokinase-type) genes were fusion proteins corresponding to the different parts of the molecule. The patients’ clinical features susceptible to the pathogenic effects of both PF and PV autoantibodies. These results suggest that were similar to those of anti-01α3 CP cases. Our present findings add the 01β3 chain of laminin- none of these proteinases are required for experimental PF and PV. Since tPA and uPA share 5 to the list of auto-antigens in CP, which thus far has included the 01α3 chain of laminin-5, substrate specificity and could compensate for each other in vivo, mice lacking both tPA and uPA BP180 and a 168-kDa antigen. This study further suggests for an important role of 01β3 chain of genes should be tested for susceptibility to PF and PV once these animals become available. laminin-5 in physiological stability of dermal–epidermal junction.

563 564 Compound Heterozygosity of a One Amino-acid Insertion/Nonsense Mutation in the Plectin E1A Represses Tissue Factor Activity in Highly Metastatic Melanoma Cell Lines Gene Associated with Autosomal Recessive Epidermolysis Bullosa with Muscle Involvement C. Voigtla¨nder, A. Rand, M. Pittelkow and M. Getz J. Bauer, F. Rouan,. B Kofler, W. Muss, R. Hametner, A. Huber, G. Pohla-Gubo, G. Wiche, J. Department of Biochemistry and Molecular Biology; and Department of Dermatology, Mayo Uitto and H. Hintner. Clinic, Rochester, Minnesota Austria; Department of Dermatology, Children’s Hospital, Institute of Pathology, General Hospital Recently we demonstrated that adenovirus E1A 12S protein is able to repress TF activity in Salzburg; Department of Dermatology and Cutaneous Biology, Jefferson Medical College, AKR-2B fibroblasts (Felts et al. Oncogene 1997, 14:1679–1685). Since it has been shown that Philadelphia, PA; Institute for Cell Biology, Vienna Biocenter, University of Vienna, Austria tissue factor (TF) activity contributes to the metastatic potential of human melanoma cell lines, Autosomal recessive epidermolysis bullosa with late-onset muscular dystrophy (EB-MD) is usually we investigated the effects of E1A on TF expression and activity in murine melanoma cell lines, caused by premature termination codon mutations in the plectin gene. In only one case so far known to be strongly metastatic in mice. We therefore stably transfected a highly metastatic EB-MD was associated with a homozygous three amino-acid in-frame deletion. Using various murine melanoma cell line, M4, with an E1A expression vector. E1A expression in the subclones mutation detection strategies, including protein truncation test, heteroduplex scanning and direct was assessed by western and northern blot analysis. TF activity was measured using a functional nucleotide sequencing covering the entire coding region of the plectin gene, we disclosed chromogenic assay. The poorly metastatic melanoma cell line C10 served as control. To further compound heterozygous mutations Q1408X/1008ins3 in a 5-year old male patient with no characterize the effect of E1A transfection on the TF promoter, we performed transient detectable muscle weakness. The stop codon predicts a truncated polypeptide, and the insertion cotransfections with E1A and different promoter constructs. In our experiments the highly mutation results in an insertion of a leucine in a row of four leucines, potentially causing the metastatic melanoma cell line M4 revealed clearly increased TF activity in comparison to the destabilization of the rod domain of plectin. Immunofluorescense of skin biopsy sections with control cell line C10. Stably transfected cell lines showed markedly reduced endogenous TF monoclonal antibodies 6C6, 10F6, 5B3 directed against the rod domain of plectin resulted in activity. In addition, we demonstrate that E1A represses the TF promoter in a dose-dependent staining only with 6C6. Cultured patient keratinocytes showed staining with 6C6 and 10F6. On manner and that the AP1 sites of the TF promoter are necessary and sufficient to mediate the patient’s muscle biopsies the same staining pattern was seen. In addition, we were unable to detect inhibitory effect of E1A. We confirm that metastatic potential is correlated with TF activity in plectin by immunoblotting using all three antibodies on keratinocyte extracts. Electron microscopy murine melanoma cell lines. Furthermore, our results strongly suggest that TF activity is repressed of the skin showed reduced numbers of hemidesmosomes which were hypoplastic. In muscle, by E1A at the level of transcription and that the AP1 sites of the TF promoter are necessary and alterations of Z-lines and partially dystrophic or absent sarcolemma were found. In this patient, sufficient to mediate this effect. haplotype insufficiency in combination with a one-amino acid insertion was sufficent to cause EB-MD possibly via alteration of the secondary structure of the rod domain of plectin. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 617

565 566 Analysis of T-Cell Receptor Gene Rearrangement for Predicting Clinical Outcome in Patients Human Papilloma Virus as Co-Factor in Non-Melanoma Skin Cancer of Immunocompetent with Mycosis Fungoides: A Comparison of Southern Blot and Polymerase Chain Reaction Methods Patients C. Haycox, T. Juarez, . DE Sabath, B. Wood, G. Wood, D. Hanke and J. Olerud A. Rust,* R. McGovern,* D. Persing and M. Pittelkow Departments of Medicine (Dermatology) and Laboratory Medicine, University of Washington, Departments of Dermatology, Biochemistry and Molecular Biology and *Department. of Laboratory Seattle, Washington; and Department of Dermatology, Veterans Affairs Medical Center, Cleve- Medicine and Pathology, Mayo Clinic, Rochester, Minnesota land, Ohio Human papillomaviruses (HPVs) have been strongly associated with the development of cervical In patients with mycosis fungoides (MF) it has been shown that the detection of a T-cell receptor intraepithelial neoplasia and cervical cancer. HPV have also been found in both skin and oral gene rearrangement (TCR-R) by Southern blot (SB) in lymph node DNA is a marker for a poor tissues of premalignant dysplasia or carcinoma. HPV has been found in a high percentage of skin prognosis and a reduced probability of survival. The purpose of this study was to determine if the samples from immunosuppressed patients, many of whom are at risk for development of use of polymerase chain reaction (PCR) would detect clonality with an increased frequency and nonmelanoma skin cancer (NMSC). HPV may therefore be a cofactor in the development of whether this would correlate to an improvement in predicting the clinical outcome in patients NMSC. With standard and nested polymerase-chain-reaction (PCR) and sequencing methods, with MF. DNA was extracted from the lymph nodes of 55 patients with MF whose outcomes we detected HPV DNA in 42.6% of 47 NMSC of immunocompetent patients. Subset analysis were prospectively evaluated. The mean period of follow up for patients still living was 9.5 y. showed HPV in 51.9% of 27 squamous cell carcinomas (SCC) and 30% of 20 basal cell carcinomas Patients were classified according to the outcome of their disease and the DNA was analyzed (BCC) as well as in 40% of precancerous actinic keratoses (AK). Normal skin harbored HPV in using SB for the 01β-receptor rearrangements and two different PCR methods for detecting 28.6% of samples. In sun-exposed, normal skin (n ϭ 9), 57.1% of samples demonstrated HPV rearrangements in the 01γ-receptor (denaturing gradient gel electrophoresis (DGGE) and capillary DNA. No nonsun-exposed normal tissue (n ϭ 5) samples harbored HPV. Initial DNA sequencing electrophoresis (CE)). DGGE used two primer sets and CE used three primer sets.The clinical identified HPV types 12 and 17 as well as six novel HPV types. These results include a patient outcome was as follows: 26 had good outcomes – complete remission or partial remission; eight with multiple SCC and a history of extensive sun-exposure and UV treatment for psoriasis. Five died of causes unrelated to MF; and 22 had poor outcomes – died of disease or had progressive of nine SCC (55.6%) harbored HPV; two sun- and one nonexposed biopsies of nontumor skin lymphoma. In the 26 patients with good outcomes only two ‘‘false positives’’ were identified by were negative for HPV. Our results suggest that HPV may play a significant role as cofactor in having a TCR-R. However, in the 22 patients with poor outcomes many ‘‘false negatives’’ for the malignant conversion to NMSC not only in the immunosuppressed but also in the TCR-R were identified: eight by SB; 12 by PCR-DGGE; and nine by PCR-CE. Although immunocompetent population. PCR is generally thought to be more sensitive for detecting clonality than SB, in this study it was not better at detecting MF patients with a poor outcome. Both PCR methods gave more ‘‘false negatives’’ than SB. The addition of a third primer set resulted in detection of clonality in three additional patients and so the selection of optimal primer sets appears to be important. We conclude that detection of clonality using PCR does not improve the ability to predict a poor clinical outcome in patients with MF compared to the SB technique

567 568 Interlocus V-J Recombination in Primary Cutaneous T-Cell Lymphomas Genomic Changes of Chromosome 1p36 in Premelanoma Lesions C. P. Willers, S. N. Wagner, D. Schindler* and M. Goos M. R. Hussein, E. Roggero, M. Sun, R. Tuthill, G. S. Wood, T. Zaim and O. Sudilovsky University of Essen MedicalSchool, Department of Dermatology, Essen, Germany; *University of Case Western Reserve University, Cleveland, Ohio Wu¨ rzburg, Department of Human Genetics, Wu¨rzburg Germany An understanding of the molecular mechanisms of premelanoma lesions would provide valuable V-J (variable-joining) rearrngements occur between, as well as within, immune receptor loci, clues for the sequence of early events during melanoma tumorigenesis. Lack of any genomic resulting in the generation of hybrid antigen-receptor genes and the formation of a variety of changes in any given premelanoma lesion could imply that such a lesion would not evolve into a lymphocyte-specific chromosomal aberrations. These hybrid genes were found in low frequencies melanoma. Past cytogenetic studies using karyotypic analysis indicated that some genetic alterations in peripheral blood lymphocytes of normal individuals but show an increased incidence in patients might occur on the chromosomal region 1p36. Using PCR based microsatellite assay we previously suffering from ataxia telangiectasia and in pesticide-exposed agricultural workers. Both later groups reported microsatellite instability (MSI) and loss of heterozygosity (LOH) on this region in 56 are at increased risk for lymphoid malignancy. As etiology of primary cutaneous T-cell lymphomas cases of melanocytic dysplastic lesions (MDN) using 10 primary melanomas and two benign nevi is still a matter of discussion we analysed potential V01γ-J01β T-cell receptor interlocus as positive and negative controls, respectively, and utilizing a panel of four microsatellite loci recombination in 22 cases of primary cutaneous T-cell lymphomas and compared it with four (D1S243, D1S214, D1S160, and D1S228). In order to better refine the chromosomal localization cases of lymphomatoid papulosis, four cases of atopic dermatitis, nine cases of primary cutaneous of those changes, we extended herein our study by analyzing seven more loci (D1S2663, D1S468, B-cell lymphomas and five cases of normal peripheral blood lymphocytes. We used a highly D1S548, D1S2740, D1S2736, D1S489, and D1S508) in the same patients. MSI and LOH were sensitive nested PCR-analysis and Southern blotting techniques and quantitated our results by found at several loci both in dysplastic nevi and in malignant melanoma but not in benign nevi. seriell dilutions of template DANN in each of the 44 cases analysed. By this means we could not Nineteen dysplastic nevi (33.9%) and seven malignant melanomas (70%) exhibited MSI while detect significant differences between primary cutnaeous T-cell lymphomas and the controls eight dysplastic nevi and five malignant melanomas displayed LOH. The markers D1S243, D1S214 regarding V01γ-J01β T-cell receptor interlocus recombination. Therefore V01γ-J01β T-cell and D1S228 had the most frequent incidence of these genetic changes. In 15 cases of MDN no receptor interlocus rearrangement as a measurement of lymphocyte-specific genomic instability genomic alterations were observed with any of the primers tested. Taken together, our results does not provide insight into the pathogenesis of cutaneous T-cell lymphomas and is not a useful suggest that similar genetic alterations exist in the premelanoma and melanoma lesions. These tool to differentiate in diagnostically difficult cases. alterations may have a role in the evolution of premelanoma and their further progression to melanoma. Also, it is tempting to think that in those 15 cases where no genetic changes were detected, a malignant transformation will not occur

569 570 A Selective Cox-2 Inhibitor, Celecoxib, Reduces UV-Induced Squamous Cell Carcinoma in CUSP, a New Member of the p53 Gene Family, is Abnormally Expressed in Basal Cell Carcinoma Hairless Mice R. Dellavalle, A. Marchbank, T. Egbert, J. DeGregori, L-J. Su, P. Walsh and L. A. Lee R. Han, J. W. Schoggins, G. A,. Scott, K. N. M. Khan* and A. P. Pentland University of Colorado School of Medicine, Denver Health and VA, Denver, Colorado Department of Dermatology, University of Rochester, Rochester, New York and *Searle Co, Genetic abnormalities, either inherited or acquired, are critical to the development of basal cell Skokie, Illinois carcinoma (BCC). Abnormalities in genes of the hedgehog pathway and in p53 commonly occur Ultraviolet (UV) light is a complete carcinogen, inducing both basal and squamous cell skin in BCC, but it is likely that additional factors contribute to the development of BCC. We and cancers, This carcinogenic effect may occur in part via the production of prostaglanclins by the others have recently described a new member of the p53 family, a gene which we called CUSP. cyclooxygenase (COX) metabolism of arachiclonic acid. COX exists in two isoforms, the Initially identified as the KET gene in rats, the gene has also been named p40, p51, p63, and p73 constitutive COX-1 and inducible COX-2. Accumulating evidence implicates COX-2 and its L. CUSP is a splicing variant of KET that is the most abundant KET transcript in the skin and is ability to catalyze the production of prostaglandin E2 (PGE2) as a major contributing factor to readily detected in the nuclei of basal keratinocytes and hair follicle epithelium. CUSP is skin tumor formation. The work in progress uses a selective COX-2 inhibitor, celecoxib, to study homologous to p53 but lacks the N-terminal transactivation domain. Due to this structure, CUSP the efficiency of COX-2 inhibition in the reduction of UV light-induced skin tumor formation appears to competitively inhibit the transactivation of p53-induced genes in hairless mice. In the current study, UVA-340 sun lamps were chosen as a light source that best In order to examine the role of CUSP in the genesis of BCC, protein, RNA, and DNA were mimic the solar UVA and UVB spectrum. Hairless mice were irradiated five days a week for the extracted from Mohs surgical specimens. In addition, sections from 16 BCC were examined with total dose of 2.62 J per cm2. Cutaneous tumors appeared 8 wk after irradiation began. Histology anti-CUSP-containing sera. Twelve of the 16 BCC failed to show CUSP protein expression by showed 95% of the tumors were squamous cell carcinoma. Microsome preparation of skin IF, despite the fact that the normal epidermis and hair follicle epithelium contained within the demonstrated increased PGE2 synthetic capacity in tumors and papillomas. This was confirmed same sections did show CUSP expression. These findings were confirmed with the use of affinity- by immunohistochemistry. When 90% of the animals had at least one tumor (6 wk postinitial purified anti-CUSP antibodies. Four of these BCC which by IF did not express CUSP, along tumor formation), the mice were randomly divided into two groups so that the tumor number with their matching nonlesional skin samples, were analyzed by Western and Northern blotting. and multiplicity were the some (P Ͻ 0.31). One half of the mice were then fed a diet containing Surprisingly, CUSP protein and mRNA were readily detected in BCC and were present in 1500 ppm celecoxib. After 10 wk, the difference in tumor number and multiplicity in the drug- comparable amounts in the BCC samples compared to the paired nonlesional skin. Preliminary treated group was 56% of controls (P Ͻ 0001). The results show that the orally administered sequencing data from one BCC shows a putative mutation at amino acid 284, which replaces Phe selective COX-2 inhibitor, celecoxib, prevents new tumor formation after the onset photocarcino- with Gln. genesis. This study demonstrates the importance of COX-2 in UV irradiation-induced tumorigenesis We conclude that CUSP abnormalities, as detected by antibody binding, are common in BCC. and suggests that treatment with celecoxib may be very useful in preventing UV-incluced skin The detection of CUSP protein by western blotting but not in IF likely indicates that CUSP tumor in humans. adopts an abnormal conformation in BCC. The conformation change could be due to CUSP mutation, or due to protein-protein or protein–DNA interactions. The high frequency of CUSP abnormality in BCC may indicate that CUSP abnormalities are an additional factor contributing to the development of BCC. 618 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

571 572 Role of the p53 and PTCH Genes in the Development of Basal Cell Carcinoma Quantirication of DNA Damage Following Ultraviolet Irradiation of Human, Cutaneous Cells Y. Yao, D. Ratner, H. Zhang, M. Peacocke and H. Tsou and Tissues Department of Dermatology, Columbia University, New York, New York M. Robert, J. Legault, G. Ross and M. Rouabria The tumor suppressor genes, p53 and PTCH, have been shown to be mutated in sporadic basal Unite´ de Biotechnologie, De´partement de Chirurgie, Universite´ Laval, CHUQ-Pavillon Saint- cell carcinoma (BCC). The mutation frequency in BCC samples was approximately 40% for p53, Francois d’Assise, Que´bec, Canada and 30% for PTCH. A loss of heterozygosity on chromosome 9q, where PTCH is located, was The incidence of the skin cancer in man has been shown to be associated with the degree of demonstrated in BCC samples containing p53 mutations. In this report, we provided the first exposure to solar radiation. The formation of different types of DNA adducts under UV radiation evidence that mutations in both p53 and PTCH genes can be found in the same BCC sample. is considered a causative event in skin carcinogenesis. The major products responsible for mutagenic Two BCC samples were obtained from an 80-year-old retired welder during Mohs’ surgeries. In effect formed by UV radiation, are cyclobutane pyrimidine dimers (CPDs) and (64) photoproducts. a BCC excised from the probandı´s temple, we detected a UV-specific C01→T change at These lesions can he formed between all adjacent pyrimidine nucleotides. The aim of this work nucleotide 1726 in exon 12 of the PTCH gene, which resulted in a premature stop codon at was to develop a simple and reproducible method to quantify the UV-induced cyclobutane dimers codon 555. From the same tumor, we also detected a G01→T change at nucleotide 830 in exon and (6–4) photoproducts in human keratinocytes monolayers as well as in human skin. Human 8 of the p53 gene, which resulted in a cysteine to phenylalanine change in codon 277. In another keratinocytes and skin samples were exposed to different doses of UVA (0, 2.5, 5, 7.5 and 10 J BCC sample excised from the probandı´s neck, we detected a G01→T change at nucleotide 564 per cm2), UVB (0, 2.5, 5, 7.5 and 10 mJ per cm2) or UVC (0, 25, 50, 75 and 100 mJ per cm2). in exon 3 of the PTCH gene, which resulted in an arginine to methionine change at codon 165. Immediately after irradiation, DNA was extracted from cells and tissues. The UV-induced CPDs Sequence analysis of the p53 gene in the same tumor tissue revealed two mutations: (i) a deletion and (64) photoproducts were converted to single-strand breaks with enzymatic (endonuclease) or of C after nucleotide 327 in exon 4 resulted in a truncated p53 protein; (ii) a G01→C change at alcalin treatments. The number of 5’-hydroxyl-containing termini generated was then determined nucleotide 559 in exon 5, which resulted in a glycine to arginine change in codon 187. by T4 polynucleotide kinase labeling of alkaline phosphatase-treated DNA with 01γ-32P ATP. Interestingly, the deletion of nucleotide C occurred at the site where the C is a part of a The percentage of each type of DNA damage was expressed as the number of phosphate groups dipyrimidine sequence. Genomic DNA from the probandı´s adjacent normal tissues did not contain incorporated, and was calculated from the specific activity of the radioactive ATP and the any of the above described mutations. Using a polymorphism in exon 12 of the PTCH gene, we radioactivity incorporated per nanograrn of DNA. Our results demonstrated a dose-deperident demonstrated a loss of heterozygosity in the PTCH gene in both tumor tissues. By contrast, the induction of CPDs after UV radiation, in keratinocyte monolayers as well as in human skin. wild type and mutant alleles of the p53 gene were present in both tumor tissues. These data Indeed, at 2.5 J per cm2 of UVB, the CPDs frequency was 8%; this frequency was 33% at 10 J demonstrated the importance of mutations in both p53 and PTCH in the development of BCC. per cm2. The same tendency was observed with (64) photoproducts. This method could provide a very sensitive and specific endpoint for determining the UV-induced molecular damage in human skin (Funded by MRC).

573 574 Vascular Endothelial Growth Factor-C (VEGF-C) and its Receptors VEGFR-2 and VEGFR-3 Differential Regulation of Collagenase-1 and Ϫ3 (MMP-1 and Ϫ13) Expression by Collagen in are Expressed in AIDS-Associated Kaposi’s Sarcoma ras-Transformed HaCaT Cell Clones M. Skobe, L. Brown,* K. Tognazzi,* J. Groupman,† K. Alitalo‡ and M. Detmar K. Airola and N. Fusenig Cutaneous Biology Research Center, Massachusetts General Hospital, Charlestown, Massachusetts; German Cancer Research Center, Division of Differentiation and Carcinogenesis, Heidelberg, *Departments of Pathology and †Internal Medicine, Beth Israel Hospital, Boston, Massachusetts; Germany ‡Haartman Institute, University of Helsinki, Finland HaCaT cells and their ras-transformed clones A5 and A5-RT3 represent a model of multistage The etiology of Kaposi’s sarcoma (KS) remains a subject of dispute. Histologically, KS is skin carcinogenesis. HaCaT cells form epidermal-like structure, A5 cells benign, and A5-RT3 characterized by clusters of spindle-shaped cells that are considered to be tumor cells and by malignant tumors following transplantation onto nude mice. We have compared the regulation of prominent vasculature. Whereas spindle cells are most likely endothelial in origin, it remains collagenase-1 and Ϫ3 (MMP-1 and Ϫ13) expression in these three cell lines cultured on plastic, controversial whether they are of lymphatic or blood vascular derivation. To test the hypothesis on type I collagen gels, and in an organotypic coculture system consisting of epithelial cells grown that lymphangiogenesis factor VEGF-C and its receptors are involved in the pathogenesis of KS, on a collagen gel populated with fibroblasts. Furthermore, we have correlated the results in these we performed in situ hybridizations and immunofluorescent stainings on 10 cases of cutaneous, in vitro systems to the in vivo situation after transplantation and subcutaneous injection of the cells HIV-associated nodular KS. Endothelial cells of small blood vessels within and immediately into nude mice. The expression of collagenase-1 was induced in A5 and A5-RT3, and to lesser adjacent to the lesions labeled strongly for both, VEGFR-1 and Ϫ2, but not for VEGFR-3 extent HaCaT, cells when cultured on a collagen gel. In the organotypic coculture system with mRNA. In contrast, spindle-shaped tumor cells strongly expressed VEGFR-2 and Ϫ3 mRNA, A5-RT3 cells and fibroblasts, the expression was further upregulated both in the epithelial and but not VEGFR-1 mRNA. Immunofluorescent staining confirmed expression of VEGFR-3 in fibroblastic compartment, as detected by in situ, northern blot, and western blot hybridization. In spindle-shaped KS cells and double immunofluorescent labeling revealed its colocalization with A5-RT3 cells transplanted and subcutaneously injected into nude mice, collagenase-1 mRNA was the endothelial cell marker CD31. Importantly, VEGF-C, the ligand for VEGFR-2 and Ϫ3 was consistently detected at the tumor front in contact with dermal matrix. Collagenase-3 was found to be expressed at high levels in large vessels surrounding KS. As determined by Northern upregulated in A5 cells grown on a collagen gel, while the A5-RT3 cells expressed very little analysis, human dermal microvascular endothelial cells also express VEGF-C in culture. These collagenase-3 mRNA under the same conditions. Furthermore, the in vivo tumors were negative results suggest important paracrine functions of VEGF-C and its receptors in the formation of for collagenase-3 mRNA. Our data demonstrate that collagenase-1 and Ϫ3 are differentially cutaneous KS. VEGF-C produced by blood vessels may stimulate proliferation of KS tumor cells regulated by collagen in these cell lines, and in the malignant A5-RT3 cells the induction of through VEGFR-3 and/or VEGFR-2. Our results are in concert with the hypothesis favoring collagenase-1 by contact with collagen may play an important role in tumor invasion in vivo. lymphatic origin and/or differentiation of Kaposi’s sarcoma Furthermore, out data indicate that expression of collagenase-3 is not required for invasion of malignant squamous cell carcinomas, but the tumor can utilize either of the collagenases for efficient degradation of the interstitial matrix

575 576 Expression, Localization, and Phosphorylation of Protein 4.1 in Cultured Human Endothelial Cells Role of Wound Myofibroblasts on Reepithelialization of Skin Using In Vitro Cutaneous Three- T. Yokota, T. Matsumura, T. Shimizu, K. Sato-Matsumura, Y. Takakuwa and A. Ohkawara Dimentional Model Department of Dermatology, Hokkaido University School of Medicine, Sapporo; Department of V. Moulin, F. A. Auger, D. Garrel* and L. Germain Biochemistry, Tokyo Womens Medical College, Tokyo, Japan Laboratoire des Grands Bruˆle´s/LOEX, Hoˆpital du Saint-Sacrement, and De´partement de chirurgie, Protein 4.1 is a major component of the erythrocyte membrane skeletal network, which plays Universite´ Laval, Que´bec; *Hoˆtel-Dieu de Montreál, Montreál, Canada important roles in maintaining membrane structure via stabilization of spectrin-actin linkages. The In human skin, wound surfaces decrease using two phenomena: reepithelialization and neodermal purpose of the present study was to show the expression, localization, and phosphorylation of formation. Numerous studies report the role of cell–cell interactions between keratinocytes and protein 4.1 in cultured human endothelial cells. Human umbilical vein endothelial cells were fibroblasts but the action of myofibroblasts on keratinocytes is not clear even though these two cultured until confluency, when the central portion of the monolayer was mechanically scraped- cell types coexist during wound healing. In this study, we investigated the influence of myofibroblasts off to allow the cells to migrate into the acellular area. The migrating cells and the confluent cells on keratinocyte growth and differentiation using a three-dimensional in vitro model of skin An were stained for protein 4.1, F-actin, and phosphotyrosin using immunofluorescent technique. histological and immunological study was performed to determine the speed and quality of To show tyrosin-phosphorylation of protein 4.1, cell lysate from confluent or nonconfluent epithelialization. When the dermis was populated with fibroblasts, a continuous epidermis was cells was immunoprecipitated with antiprotein 4.1 antibody, separated by SDS-PAGE, and formed within 7 d. In contrast, when myofibroblasts were present in collagen gels, the complete immunoblotted using antiphosphotyrosin antibody. Confocal laser scan microscopy revealed that reepithelialization never occurred within the 10-day period studied and an epidermis more protein 4.1 and phosphotyrosin were colocalized at the tips of actin filaments in the migrating disorganized can be observed. An immunohistochemistry study showed a similar expression of cells, whereas only diffuse cytoplasmic staining of protein 4.1 and intercellular staining of transglutaminase and vimentin when myofibroblasts and fibroblasts were present in dermis. In phosphotyrosin were observed in the confluent cells. Phosphorylation of protein 4.1 was also tissue containing myofibroblasts, laminin and collagen IV appearance was delayed compared to confirmed by immunoprecipitation. The presence of phosphorylated protein 4.1 at the tips of fibroblast tissues. In the same way, fibronectin deposition by keratinocytes at the dermo–epidermal actin filaments in the migrating cells, but not in the confluent cells, suggested that protein 4.1 is junction was completely inhibited when myofibroblasts were present. In vitro, the presence of involved in cell–matrix interactions of endothelial cells. myofibroblasts in the dermis induced a delay of epithelialization and a disorganization of epidermis not seen in the presence of fibroblasts which are known to be required for a good keratinocyte growth. So, we concluded that both cell types have various actions on keratinocytes probably through growth and differentiation factor production. These results also suggest that myofibroblasts are different and not sufficient to promote keratinocyte growth. We consider that this model is a good tool to further understand myofibroblast–keratinocyte interactions and, more generally, to define intercellular relations involved in cutaneous wound healing. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 619

577 578 In Vitro Assembly of Mouse Hard 01α-Keratins into Intermediate Filaments A Combined Ultraviolet B Light and Electromagnetic Field Exposure System to Modify Epidermal H. Wang, L. Bellincampi, L. Jones* and P. Steinert and Lymphocyte Cell Growth and Development Laboratory of Skin Biology, NIAMS, NIH, Bethesda, Maryland; and *Division of Wool W. Balcavage, G. Nindl, D. Spandau and J. Swez Technology, CSIRO, Belmont, Australia Terre Haute Center for Medical Education, Indiana University School of Medicine, Terre Haute, Many details of the assembly and structure of cytokeratin intermediate filaments are now well Indiana; Department of Dermatology, Indiana University School of Medicine, Indianapolis, understood. However, much less is known about the hard 01α-keratin filaments. In particular, Indiana; and Physics Department, Indiana State University, Terre Haute, Indiana the inability to assemble their constituent chains into filaments in vitro has hampered analyses of In psoriatic lesions, the normal program of epidermal differentiation is dramatically altered with their structures. To begin to resolve this issue, the mouse hair keratin MHKA-1 and MHKB-2 the epidermis becoming hyperproliferative due to inappropriate release of cytokines from chains were chosen as the obligatory Type I and Type II hard 01α-keratin partners. First, the two chronically activated inflammatory lymphocytes. Selective elimination of these inflammatory cells full-length cDNAs were generated by PCR using mouse hair follicle RNA, followed by cloning would be therapeutically beneficial and we are developing equipment and methods to test the into the pET11a expression vector system. Following induction, keratins were expressed in yields feasibility of this idea. Based on preliminary observations we hypothesized that exposure to of Ͼ1 mg per ml, recovered from inclusion bodies by dissolving with a buffer of 8 M urea combined electromagnetic fields (EMF) and UV light can selectively eliminate inflammatory containing 50 mM Tris-HCl (pH 8.8), 2 mM EDTA and 5 mM DTT, and purified by lymphocytes in psoriatic epidermis. In tests of this hypothesis we have shown that weak EMFs chromatography on a MonoQ ion exchange column. Equimolar amounts of the Type I and II activate T-cell receptors in a lymphocyte cell model (Jurkat E6–1 cells) exposed to EMFs in a chains were mixed in 9.5 M urea, and dialyzed through reduced urea concentrations of 6 M, 2 Merritt coil powered by a B & K signal generator and a Kepco amplifier. The EMF on a plane M and 0 M urea in degassed buffers of many compositions. We found that optimal assembly into through the center of the coil was shown to be homogeneous (Ϯ5%) within a 12-cm3 volume filaments occurred in a buffer of 10 mM Tris-HCl (pH 6.5), 150 mM NaCl, 5 mM MgCl2 and in the center of the coil system. At a centrally located point, the EMF was linear with applied 5 mM DTT. When examined by negative staining, filaments were about 10 nm diameter and up voltage up to about 2 mTesla. Using this system, an EMF of 0.1 mTelsa inhibited Jurkat cell to 1 µm long. Thus hard 01α-keratins can be induced to assemble in vitro in good yields. growth and induced cell death. Using DNA-based flow cytometry, assays of caspase-3 activation, and chromosomal DNA fragmentation, we concluded that the EMF induced Jurkat cell death was due to apoptosis. A UV lamp mounted in a non ferromagnetic holder was incorporated into the coil system with the transformer located remotely. In independent experiments, we showed that low doses of UVB irradiation do not affect human keratinocyte growth or proliferation. These UV and EMF based observations support the hypothesis that weak noncarcinogenic EMFs can synergistically reduce the dose of UVB radiation required to induce lymphocyte apoptosis and possibly stimulate FasL related apoptosis by inducing additional lymphocyte Fas or keratinocyte FasL. We anticipate the combined effects of EMFs and UV light to be synergistic and provide the basis for further development of new therapeutic modalities for psoriasis and other inflammatory epidermal diseases.

579 580 Human Periplakin: Genomic Organization in a Clonally Unstable Region of Chromosome 16p Role of Homeobox Transcription Factor Dlx3 in the Regulation of the Trichohyalin Gene with an Abundance of Repetitive Sequence Elements K. Wu, S. Jang, P. Steinert, J. Bryan and M. Morasso S. Aho, K. Rothenberger, E. Tan, Y. Ryoo, B. Cho, W. McLean and J. Uitto Laboratory of Skin Biology, NIAMS, NIH, Bethesda, Maryland Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Philadelphia, Trichohyalin (THH) is an intermediate filament-associated protein that is synthesized in the Pennsylvania medulla cells and inner root sheath (IRS) of the hair follicle. Serial deletion and mutational analysis Periplakin, a member of the plakin family of proteins, has been recently characterized by cDNA of a proximal promoter of the THH gene have determined cis-acting elements that confer tissue- cloning, and the corresponding gene, PPL, has been mapped to human chromosome 16p13.3. specific and differentiation-specific expression to the THH gene. We show the promoter of THH Periplakin has also been shown to serve as an autoantigen in a malignancy associated autoimmune includes a AT-rich motif with homology to the Dlx3 consensus binding site. Mobililty shift blistering disease, paraneoplastic pemphigus. In this study, we have elucidated the intron-exon experiments showed binding of the AT-rich-site with recombinant Dlx3 protein. Super-shift organization of human PPL and characterized its promoter region. The flanking 5Ј sequences analysis with specific antibody against Dlx3 showed that Dlx3 participates in the formation of were rich in G and C (~80%) and included multiple AP2 sites and a SP1 site, while no canonical complexes between proteins in hair follicle nuclear extracts and the AT-rich binding site. Previously, TATA or CCAAT sequences were found. The functionality of the upstream sequences (–709 to a Dlx3 binding site was reported in the proximal promoter region of the granular late differentiation ϩ135) as a promoter in cultured epidermal keratinocytes was detected by a CAT reporter gene, gene profilaggrin (PF). Interestingly, the Dlx3 binding sites in the THH and PF genes are closely and a limited region (–382 to ϩ135) showed activity in cultured dermal fibroblasts, attesting to located to the TATA box. The Dlx3 motif is 6 bp upstream of the TATA in the THH promoter cell-type specificity of the promoter. The genomic organization, including the intron-exon borders, and 4 bp downstream of the TATA in the PF promoter. Using site direct mutagenesis, we have was determined by direct nucleotide sequencing of human genomic P1 clones. Comparative switched the TATA-Dlx3 motifs between the PF and THH creating chimeric promoters to analysis of cDNA and genomic sequences revealed that PPL consists of 22 exons, the distribution determine the effect of the spatial arrangement on tissue-specific expression and/or promoter of exons in PPL being consistent with other plakin genes: 21 small exons, separated by large activity. Transient transfection of the chimeric promoters showed that for THH, the activity of introns, encode the amino-terminal globular domain, and one large exon encodes the entire rod the chimeric promoter was 4-fold higher than the wild type promoter, while for PF, the expression and the tail domains. Characterization of four P1 clones spanning the PPL locus revealed multiple was 3-fold lower than the wild type. These results indicate that the arrangement between the Alu repeats, 20 of them within 33 kb of the entirely sequenced segments (0.60/kb), in addition Dlx3 and TATA box motifs is important for the proper role of Dlx3 in the regulation of to numerous MIR and L1 elements. These repetitive elements could lead to the clonal instability profilaggrin in the interfollicular epidermis and in THH in the hair follicle. detected throughout the genomic P1 clones and may give rise to the genomic rearrangements possibly underlying the paraneoplastic pemphigus.

581 582 01α-Melanocyte-Stimulating-Hormone (01α-MSH) induces Histamine Release from Human Demonstration of an In Vivo Imaging System which is Capable of Obtaining Images and Spectral Skin Mast Cells In Vitro Information from Skin and Follicles Non-Invasively A. Gruetzkau, M. Artuc, T. Luger* and B. Henz J. Sullivan, K. Yuan, M. Aronson and H. Knaggs Department of Dermatology, Charite´, Campus Virchow Clinic, Humboldt-University, Berlin, Unilever Research, Edgewater, New Jersey *Department of Dermatology, University Muenster, Germany The aim of this work was to develop a system capable of imaging low levels of fluorescent light The aim of the present study was to explore novel neuropeptide-mediated, IgE-independent from skin and obtaining spectral information about the fluorescence. Such a system would be mechanisms leading to mast cell activation. For this purpose, we investigated the influence of very powerful for in vivo skin imaging and would have a variety of applications. A noninvasive 01α-MSH on the release of histamine in human skin mast cells. Additionally, we studied the in vivo imaging system has been designed which includes a light source capable of producing expression of melanocortin receptors (MC-1R and MC-5R) in mast cells at the mRNA-level by monochromatic light, an air-cooled slow scan CCD camera and a liquid crystal tunable filter. The RT-PCR-techniques and at the protein-level by FACS-analysis. Mast cells were immunomagnet- system has the capability of acquiring images of the skin, obtaining both the excitation spectra ically enriched up to 90% from normal human foreskin by positive selection using an anti-CD117 and emission spectra. The system has been used to measure the fluorescent spectra for follicles monoclonal antibody and a MiniMACS separation device. 01α-MSH induced a dose-dependent giving reproducible results. Two emission peaks were found. One was the orange-red porphyrin increase in histamine release at a concentration range of 0.01–1.0 pM. This highly specific response fluorescence peak with a maximum excitation at 405–415 nm and a maximum emission peak at suggests receptor-mediated effect, an assumption that was supported by RT-PCR experiments: 605 nm. This spectral measurement agrees well with the literature. Another emission peak in the Skin mast cells and the human leukemic mast cell line-1 (HMC-1) expressed mRNA for the yellow-green 560 nm region was also found, with a peak excitation of 470–475 nm. Currently, MC-1R and HMC-1 cells additional mRNA for the MC-5R. At the protein level, using receptor- further investigation is required before the fluorophore causing this peak can be identified. In specific antisera, MC-5R was shown to be expressed in HMC-1 as well as in skin mast cells, with addition, a skin autofluorescence emission peak at 500 nm was identified, although this varied only low level expression of MC-1R in both cell types. Taken together, these results suggest that depending upon the skin type. The difference in autofluorescence intensity is best explained by 01α-MSH may modulate mast cell activation via constitutively expressed melanocortin receptors. the differences in distribution and amount of melanin between the subjects. We have demonstrated a non invasive imaging system which can acquire images of the skin, in real time, in vivo. Both spectral and spatial distribution of data can be obtained through the use of this system. 620 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

583 584 Motile Properties of Keratin Intermediate Filament Network Resveratrol, a Phytoalexin, is a Potent Modulator of Epidermal and Dermal Function K. Yoon,*† M. Yoon,* R. Moir,* P. Steinert,‡ M. Omary‡ and R. Goldman* R. Carson, K. Patel and S. Pillai *Department of Cell and Molecular Biology, North-western University, Chicago, Illinois; Unilever Research, Edgewater, New Jersey †Department of Dermatology, Yonsei University, Seoul, Korea; ‡Laboratory of Skin Biology, Resveratrol is a phytoalexin found in plants such as peanuts, pines and grapes. Biological activities NIH, Bethesda, Maryland; §Department of Medicine, VA Palo Alto Health Care System, Palo attributed to this compound include: cancer chemopreventive, antiatherosclerotic, antioxidant, Alto, California inhibition of platelet aggregation, induction of differentiation of leukemia cells, and inhibitor of We have made cDNA constructs encoding green fluorescent protein (GFP)-keratin 14 and GFP- eicosanoid biosynthesis. In this study, we evaluated the effects or resveratrol on epidermal and keratin 18 to study the dynamic properties of keratin intermediate filaments (IF) in live epithelial dermal function. Resveratrol was a potent inhibitor of keratinocyte proliferation. Half maximal cells. Following transfection, fluorescence microscopy reveals that GFP-keratin is incorporated concentration of inhibition was 1 µM; complete inhibition of proliferation was achieved at 25 into keratin IF networks in PtK2, A431, and HeLa cells. Reorganization of GFP-keratin IF during µM. Inhibition was reversible indicating that resveratrol was nontoxic to keratinocytes. Ten µM mitosis in living PtK2 and HeLa cells appears exactly the same as in fixed and stained cells. Time- resveratrol significantly stimulated the expression of keratinocyte specific transglutaminase-1. lapse observations show that tonofilaments exhibit bending and kinking movements. Kinks can Keratinocytes grown in the presence of 1–10 µM resveratrol in low calcium medium stratified propagate along tonofilaments and in some cases they completely disappear. Short filamentous into multilayered islands displaying morphological features of differentiation. Resveratrol had no structures with two free ends, keratin squiggles, are found in the cell periphery, about 15.5% of effect on the proliferation of fibroblasts. Ten µM resveratrol stimulated the GAG synthesis of which translocate at an average rate of 0.24 µm per min. Fluorescence recovery after photobleaching dermis of pig skin organ cultures. These studies suggest that resveratrol, a polyphenolic compound (FRAP) analyses show that bleach zones across GFP-tonofilaments generally recover their present in nature, induces epidermal differentiation and dermal matrix synthesis, and may be useful fluorescence within 3 h in living PtK2 cells. The fluorescence recovery rates are similar with both as a safe and effective therapeutic agent for skin disorders. GFP-keratin 14 and GFP-keratin 18 with the recovery half-time (t1/2) of 107 min. Frequently, bleach zones move during fluorescence recovery at an average rate of 0.06 µm per min. The effects of nocodazole or cytochalasin-B on the rates of the movements are under investigation. Our results show that keratin IF networks are highly dynamic and exhibit motile properties in live cells.

585 586 A Role for p53 in Regulation of Fibronectin and Integrin Expression The Sodium/Hydrogen Antiporters (NHE) in Epidermal pH Regulation J. A. Bush, V. Ho and G. Li M. Behne, Y. Oda, W. M. Holleran and T. Mauro Department of Medicine, Division of Dermatology, University of British Columbia, Vancouver, Department of Dermatology, UC San Francisco & VA Med. Center, San Francisco, California BC, Canada The external layer of the epidermis, the stratum corneum (SC), shows an outward-directed, The tumor-suppressor gene, p53, is known to play critical roles in suppression of the transformed increasingly acidic pH. Neither the origin nor the function of this ‘‘acid mantle’’ in both normal phenotype, progression, malignancy, and metastasis. We have observed two unusual phenotypes cutaneous function and/or disease are understood. We recently showed that barrier recovery of dermal fibroblasts (Fbs) isolated from p53–/– mice not evident in p53ϩ/ϩ controls: the capacity proceeds normally at an acidic pH, whereas it is delayed at neutral pH (i.e. pH 7; Arch Derm Res to grow in soft agar; and, detachment from plastic cultureware postconfluency. Since fibronectin 290:215, 1998). Although lamellar body secretion is unimpeded at neutral pH, the postsecretory, (FN) and its receptor integrin, the 01α501β1 heterodimer, play important roles in transformation, extracellular processing of lipids appears disturbed. This result is consistent with the known acidic angiogenesis, and metastasis, we investigated whether p53 suppresses the transformed phenotype pH optima for enzymes responsible for lipid hydrolysis in the SC, e.g. 01β-glucocerebrosidase. by regulating the expression of FN and/or integrin 01α501β1. We found that the expression of We now have determined that sodium-hydrogen exchanger proteins (NHE) are present and active integrin subunits 01α5 and 01β1 is reduced in p53–/– Fbs harvested at progressively higher in cultured human keratinocytes (CHK), and propose that these antiporters may play a role in the confluency. Furthermore, we found that FN transcripts are reduced in p53–/– Fbs compared to generation of the acidic environment of the SC. Western Immunoblots revealed NHE3 in both p53ϩ/ϩ controls upon confluency. Taken together, these results suggest that p53 is an upstream human epidermis and CHK. Increased NHE3 levels were observed in postconfluent CHK, regulator of both FN and 01α501β1. Therefore, p53 may suppress cell transformation and suggesting that NHE3 expression is enhanced as CHK differentiate. Conversely, as numerous metastasis by upregulating the expression of FN and integrin 01α501β1. nonspecific bands were obtained with the antibodies currently available, the presence of NHE1 could not be definitely identified on Western immunoblots. However, the expression of NHE1 mRNA in both CHK and epidermis was evident by PCR. Finally, functional studies with the pH sensitive dye BCECF revealed that CHK can recover from an acid load (i.e. export Hϩ), a recovery that is blocked by low-dose (10–6 M)amiloride suggesting that intracellular pH is controlled by (an) NHE. Together, these data demonstrate: (i) the presence of both NHE1 and NHE3 in human keratinocytes; (ii) that NHE3 levels increase in more differentiated cells, consistent with a proposed role in acidifying the SC extracellular space; and (iii) that functional NHE are present in both keratinocytes and mammalian epidermis and may play a key role in regulating intracellular and extracellular pH, although the precise NHE(s) responsible for these acidification processes have not yet been delineated.

587 588 Oxysterol Activators of LXR Induce Differentiation and Inhibit Proliferation in Human Ker- Keratinocytes Expressing Increased Level of Functional Fas Ligand Induce Apoptosis in Cocultured atinocytes T-Cells K. Hanley, D. Ng, S. He, P. Lau, K. Min, P. Elias, D. Bikle, D. Mangelsdorf, M. Williams and R. Arnold, M. Seifert, K. Asadullah* and H. Volk K. Feingold Departments of Medical Immunology and *Dermatology, Humboldt-University of Berlin, Charite´, Departments of Dermatology, Medicine, Pediatrics, University of California, San Francisco, Berlin, Germany California; Howard Hughes Medical Institute and Department of Pharmacology, University of Binding of Fas Ligand (CD95L, FasL) by its counterreceptor Fas (CD95) induces apoptosis in a Texas SW Medical Center at Dallas, Dallas, Texas, and the Dermatology and Medical Services, wide variety of target cells. Apoptotic keratinocytes and lymphocytes expressing Fas were observed Department of Veterans Affairs Medical Center, San Francisco, California in the lesional epidermis of many inflammatory skin diseases. Therefore, we hypothesized whether Activators of nuclear hormone receptors are important regulators of epidermal development and FasL might be expressed by keratinocytes in a cytokine dependent fashion and whether it might differentiation. We showed previously that naturally occurring fatty acid activators of PPAR induce contribute to the observed apoptotic events. For this purpose we analyzed primary human keratinocyte differentiation. Here we asked whether oxysterols, another class of lipids formed de keratinocytes and cells of the transformed keratinocyte cell line HaCaT. Keratinocytes grown to novo in the epidermis and which activate LXR, regulate human keratinocyte differentiation. confluency were exposed to a variety of cytokines and thereafter FasL expression and apoptosis mRNA and protein levels of involucrin (INV) and transglutaminase (TG), protein markers of was determined by FACS- and immunoblot analysis. We observed that keratinocytes exposed to differentiation, increased 2–3-fold in keratinocytes incubated in the presence of 25- or 22R- proinflammatory cytokines (IFN-01γ, IL-1β, TNF-01α) showed a significant increased FasL hydroxycholesterol, both under low and high calcium conditions. Furthermore, rates of cornified expression pattern. This upregulation was dependent on new protein synthesis and counterregulated envelope formation, an indicator of terminal differentiation, also increased 2-fold with oxysterol by anti-inflammatory cytokines (IL-10, TGF-01β1). Enhanced cell surface FasL expression induced treatment. In contrast, the rate of DNA synthesis was inhibited approximately 50% by oxysterols. apoptotic events within the generated keratinocyte-monolayer as well as in cocultured Fasϩ Jurkat Activity of a TG-or an INV-promoter-luciferase construct transfected into keratinocytes increased T-cells. In addition, genetically engineered keratinocytes expressing an enhanced amount of FasL approximately 2–3-fold following oxysterol treatment. Deletion of the region containing –2452 generated stable monolayers which induced apoptosis in cocultured Fasϩ T-cells. Preliminary skin to –1880 bp of the INV gene, or mutation of the AP-1 site located within this region (–2116 transplantation experiments performed in an allogeneic rat model suggest that an enhanced to –2110 bp), resulted in loss of oxysterol responsiveness. Finally, we demonstrate the presence of expression of FasL on skin allografts might be responsible for a prolonged skin graft survival as it LXR01α and 01β mRNA in keratinocytes and show that oxysterols stimulate expression of an was hypothesized for immune privileged sites (eye, testis). In summary, our data suggest that LXR response element transfected into keratinocytes. These results indicate that oxysterols induce cytokine activated keratinocytes expressing an enhanced level of FasL might play a central role in keratinocyte differentiation, inhibit proliferation, and stimulate TG transcription, and specifically the cell turn-over during inflammatory skin diseases. Furthermore, an enhanced FasL expression AP-1-dependent INV transcription, effects that may be mediated by LXR. pattern on genetically engineered skin transplants might protect them for skin infiltrating cytotoxic T-cells. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 621

589 590 α Presence of Cathepsin B-Like Enzyme in Cultured Human Keratinocytes Zinc-01 2-Glycoprotein Modulates the Adhesion and Proliferation of Oral Tumor Cells T. Ohno, D. Ueda, Y. Takata and S. Dekio M. M. Brysk, G. Lei, I. Arany and S. K. Tyring Department of Dermatology, Shimane Medical University, Izumo, Japan Depts. of Dermatology and of Microbiology & Immunology, University of Texas Medical Branch, Cathepsin B (CB) is an SH proteinase present in the ubiquitous mammalian cells. It can be Galveston, Texas α expected that there is a CB in human keratinocytes also. However, presence and physiology have Zinc-01 2-glycoprotein (ZAG) is a soluble protein found in most body fluids and in a wide little been known. In the present study, we proved that CB is present in human keratinocytes. variety of glandular epithelia; we have also found it in stratified epithelia. ZAG expression decreases The CB in the cultured human keratinocytes was tried to be stained with antibody to the CB of with tumor dedifferentiation in oral and epidermal tumors. Its sequence has considerable homology the human hepatocytes. Since after the washing with a buffer, the antibody remained in the with major histocompatibility complex (MHC) class I01α chains. We have cloned ZAG from cytoplasm diffusely, CB was proven to be surely in the keratinocyte cytoplasm. Next, we asked epidermal keratinocytes and expressed the recombinant protein in a Baculovirus expression system. on the movement of the CB in human keratinocytes by calcium ion. As the result, the CB in ZAG contains an RGD (Arg-Gly-Asp) sequence, in common with most extracellular matrix the cytoplasm of keratinocytes was moved dramatically to the cell membrane of the keratinocytes proteins. In this study, we characterize the adhesive properties of ZAG and compare them to cultured in the medium containing high dose of calcium ion. These results indicated that the CB those of other common matrix proteins. Tu-138 oral SCC cells bind and spread on ZAG in a may play an important role in differentiation of human keratinocytes. dose-dependent fashion, as determined by phase-contrast microscopy and quantitatively by MTT assay. Cell attachment to a ZAG matrix is inhibited in a dose-dependent manner by the synthetic peptides RGD, RGDS, and RGDV. Attachment and spreading are also inhibited by an antibody α β α β α β to integrin 01 501 1, but not by antibodies to several other integrins (01 v01 3,01 301 1, and α β 01 201 1), implying that cell attachment occurs primarily through the fibronectin receptor. Tu- 138 cells plated on a ZAG matrix were also growth-inhibited. No similar inhibition was obtained when the same cells were grown on other matrix proteins (fibronectin, vitronectin, laminin, collagens I and IV). We believe that ZAG plays a role in tumor differentiation and suppression

591 592 α Zinc-01 2-Glycoprotein Modulates the Adhesion and Proliferation of Oral Tumor Cells Regulation of Annexin I Expression and Localization by Phorbol Ester, Epinephrine, Hydrocortis- M. M. Brysk, G. Lei, I. Arany and S. K. Tyring one, and Calcium in Cultured Human Keratinocytes Departments of Dermatology and of Microbiology and Immunology, University of Texas Medical K. Sato-Matsumura, T. Matsumura, H. Koizumi, H. Sawa, K. Nagashima and A. Ohkawara Branch, Galveston, Texas Department of Dermatology and Molecular and Cellular Pathology, Hokkaido University School α Zinc-01 2-glycoprotein (ZAG) is a soluble protein found in most body fluids and in a wide of Medicine, Sapporo, Japan variety of glandular epithelia; we have also found it in stratified epithelia. ZAG expression decreases Since annexin I was reported as a component of cornified envelope, it has been suggested that with tumor dedifferentiation in oral and epidermal tumors. Its sequence has considerable homology annexin I plays an important role in the process of keratinization. To elucidate the function of with major histocompatibility complex (MHC) class I01α chains. We have cloned ZAG from annexin I in keratinization, we investigated the effect of TPA, epinephrine, hydrocortisone, and epidermal keratinocytes and expressed the recombinant protein in a Baculovirus expression system. calcium on the expression and localization of annexin I in cultured human keratinocytes. Normal ZAG contains an RGD (Arg-Gly-Asp) sequence, in common with most extracellular matrix human keratinocytes were cultured in serum free culture medium (0.15 mM calcium) until 70% proteins. In this study, we characterize the adhesive properties of ZAG and compare them to confluency. After incubation with TPA (100 nM), epinephrine (50 µM), hydrocortisone (10 µM), those of other common matrix proteins. Tu-138 oral SCC cells bind and spread on ZAG in a or calcium (1.8 mM) for 24 h, cells were examined using immunofluorescence, western blot, and dose-dependent fashion, as determined by phase-contrast microscopy and quantitatively by MTT northern blot analysis. Immunofluorescent microscopy showed that incubation with TPA or assay. Cell attachment to a ZAG matrix is inhibited in a dose-dependent manner by the synthetic calcium increased the membranous staining of annexin I, whereas incubation with epinephrine or peptides RGD, RGDS, and RGDV. Attachment and spreading are also inhibited by an antibody hydrocortisone did not. Increase of annexin I on the cell membrane was also confirmed by α β α β α β to integrin 01 501 1, but not by antibodies to several other integrins (01 v01 3,01 301 1, and western blot, which showed increased annexin I in the cell membrane fraction, but not in the α β 01 201 1), implying that cell attachment occurs primarily through the fibronectin receptor. Tu- cytosol fraction, of the lysates taken after incubation with TPA or calcium. On the contrary, 138 cells plated on a ZAG matrix were also growth-inhibited. No similar inhibition was obtained incubation with epinephrine or hydrocortisone decreased annexin I in the cell membrane fraction. when the same cells were grown on other matrix proteins (fibronectin, vitronectin, laminin, Interestingly, incubation with TPA, but not with calcium, upregulated mRNA level of annexin collagens I and IV). We believe that ZAG plays a role in tumor differentiation and suppression I. Because TPA and calcium are the agents known to promote keratinization, our data suggested that annexin I expression on the cell membrane was closely related to the process of keratinization.

593 594 The Effect of Dibutyryl Cyclic AMP on Wound Healing and Expression of Basement Mem- Differentiation-Associated Localization of the Mixed Lineage Kinase ZPK in Normal Human Skin brane Protein L. Germain, J. Fradette, R. Guignard, G. Grondin* and R. Blouin* H. Onuma, C. Matsui and M. Morohashi Laboratoire d’Organogeńe`se Expe´rimentale, Hoˆpital du Saint-Sacrement and De´partement de Department of Dermatology, Toyama Medical and Pharmaceutical University, Toyama, Japan Chirurgie, Universite´ Laval, Que´bec, Canada, and *De´partement de Biologie, Faculte´ des Sciences, The effect of dibutyryl cyclic AMP (dbcAMP) on keratinocyte division has been investigated and Universite´ de Sherbrooke, Que´bec, Canada thought to accelerate wound healing. We have tried to elucidate the effect of dbcAMP cutaneous ZPK is a recently described protein serine/threonine kinase which is classified as a member of the wound in two-dimensional observation. After making surgical wound on the back, hairless mice mixed-lineage (MLK) family of protein kinases. Previous in situ hybridization studies of ZPK gene were treated with dbcAMP ointment or control. Epidermal sheet was made from both groups of expression have been carried out for embryonic and adult mouse tissues (Blouin et al. DNA and mice by 1 M sodium bromide treatment after bromodioxyuridine(BRDU) injection to label Cell Biology, 1996, 15:631–642) and the overall pattern of expression suggests that ZPK could be dividing cells. In re-epithelizating area of dbcAMP treated skin wound, BRDU labeling index of involved in the regulation of epidermal cell proliferation and differentiation. In order to get more basal keratinocyte were statistically higher than control during wound healing. To study other insights about the function of this new kinase, we looked at its localization in normal human skin. action of dbcAMP in wound healing, we investigated expression of laminin5 (Lam5), basement ZPK expression was investigated at the mRNA and protein levels by in situ hybridization, western membrane zone (BMZ) protein that is crucial for keratinocyte migration and hemidesmosome blotting and indirect immunofluorescence studies. Our results reveal that ZPK has a very precise formation, in same tissue immunohistochemically. On fifth day after wound, Lam5 deposit in pattern of expression in skin epithelia as both mRNA and protein are restricted to the granular BMZ of wound edge was increased compare to control. The present results suggest that increased layer of epidermis and inner root sheath of hair follicles. Immunofluorescence detection of ZPK Lam5 expression by dbcAMP might enhance keratinocyte migration and cutaneous wound close. on skin from various body sites and donors of different ages always showed ZPK expression at The study regarding the expression of Lam5 at the RNA and protein levels is in progress with this precise localization. This expression pattern of ZPK supports the hypothesis that this kinase cultured murine keratinocyte. may act as a negative regulator of cell proliferation or an inducer of terminal differentiation. 622 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

595 596 Stem Cells In Vitro: Longevity Versus Colony Forming Efficiency The Human and Mouse Sciellin Genes have a Complex Genomic Organization M. Matic,* A. Shih,* D. M. Siegel,† L. Taichman* and M. Simon*† H. Baden, M. Champliaud and P. Olson Departments of *Oral Biology and Pathology, †Dermatology, SUNY, Stony Brook Cutaneous Biology Research Center and the Department of Dermatology, Harvard Medical Homeostasis of self-renewing tissues relies on the presence of stem cells. Keratinocyte stem cells School, Charlestown, Massachusetts are long-lived, slowly cycling, endowed with high proliferative capacity, and are believed to reside The recently identified human sciellin cDNA revealed that the 75.3 kDa protein has a central in a niche, which is well protected, vascularized and innervated. It has previously been documented domain of 16 repeating units and a carboxyl domain containing a single LIM motif. The intron- that cultured human epithelia contain keratinocytes which act as stem cells following engraftment exon structure of the human sciellin gene was detemined prior to developing a patient screening onto patients, and onto immuno-incompetent mice. It is generally assumed that epidermal stem assay. In order to develop animal models to study the function of sciellin, the mouse sciellin cells have both a high colony forming efficiency (CFE) and long-term growth potential. However, cDNA and genomic structure were characterized. The 73.2 kDa mouse sciellin protein contained the association between CFE and longevity (cumulative population doublings) has never been a central domain of 15 repeating units, which differed from the human in the number and order examined directly. We examined the long-term in vitro growth potential of adult human epidermal of repeats, and a single LIM domain at the carboxyl end. The mouse and human proteins had keratinocytes as a function of CFE using more than 100 keratinocyte clones obtained from each 74% amino acid similarity over their entire lengths, but this number increased to 99% similarity of three biopsies. CFEs were determined at each passage. The correlation between long-term when only the carboxyl LIM domains were considered. A mouse embryo developmental Northern growth potential and CFE at early passage was weak. However, reduction of colony forming blot demonstrated a single band of 3.6 kb and peak expression at 17 d pc, concomitant with the efficiency to less than 1% generally predicted the onset of senescence. Twenty per cent of the differentiation of stratified squamous epithelia. The mouse gene was mapped to chromosome 14 clones from each biopsy were able to undergo an average of 57 doublings; these clones have the by FISH. Analysis of the human sciellin gene structure revealed that each repeat unit is encoded capacity to generate 1013–1018 cells. In each of the biopsies, 6%–10% of the clones were long by a separate 60 bp exon. The repeat-encoding exons were separated by introns varying in size lived and underwent more than 100 doublings; these clones have the capacity to generate more from 0.5 kb to 5.0 kb. Preliminary analysis of the mouse sciellin gene revealed a similar intron- than 1030 cells. The percentage of long-lived clones is comparable to the percentage of keratinocyte exon organization. The complex genomic structure of sciellin further supports our earlier evidence stem cells thought to be present in the basal layer of epidermis. that sciellin is unique compared to other cornified envelope precursors. Many other cornified envelope proteins are clustered in the Epidermal Differentiation Complex of human chromosome 1 and possess a simple genomic structure in which all of the protein is encoded by a single exon. Shuffling of functional domain-containing exons caused by intron-mediated recombination is thought to be an important evolutionary mechanism for the creation of new genes. This theory is consistent with what we have observed with respect to the repeat-containing and LIM domain- containing exons of the sciellin gene.

597 598 Bcl-X(L) is Upregulated in Mouse Keratinocytes Cultured in the Presence of High Calcium Medium Long-Term Keratinocyte Culture of Individual Harlequin Ichthyosis (ichq/ichq) Mice M. Kuechle, B. Hager and P. Fleckman M. Michel, D. Boggess, J. Sundberg and B. Dale Department of Medicine/Dermatology, University of Washington, Seattle, Washington Department of Oral Biology, University of Washington, Seattle, Washington, and The Jackson Terminal differentiation of keratinocytes involves expression of a distinct array of proteins followed Laboratory, Bar Harbor, Minnesota by collapse of the cell into a rigid, two-dimensional squame. The morphologic changes of terminal Harlequin ichthyosis (HI) is a severe scaling skin disorder in which infants die within few weeks differentiation share many characteristics with apoptosis. However, the biochemical relationship after birth. Therefore, a very limited amount of material is available to us for biochemical and between terminal differentiation and apoptosis remains unclear. Mouse keratinocytes, when genetical studies. A harlequin ichthyosis (ichq/ichq) mouse mutation has been described and the cultured in low calcium medium, display characteristics of the basal layer of the epidermis. When phenotype has many similarities to the human HI type 2. This mouse model is an important tool switched to medium containing high calcium, mouse keratinocytes stop dividing and express to investigate disease mechanisms of HI. We used a new mouse keratinocyte cell culture procedure protein markers of the upper layers of the epidermis, including profilaggrin and loricrin. Using to grow keratinocytes from normal, heterozygotes and affected ichq mouse. Cells were obtained this system, we examined RNA from C57/BL6 mouse keratinocytes cultured in low and high from 2-d-old mice from interspecific heterozygous matings. The mice are indistinguishable from calcium media with a panel of probes against Bcl-2 family members and the caspase family each other at that age, since the onset of the clinical phenotype corresponds with the emergence members in an attempt to clarify the relationship between apoptosis and terminal differentiation. of the hair fibers from follicles at5dofage. Individual mice were genotype using a PCR-based Within the Bcl-2 family members, mouse keratinocytes express RNA for Bcl-X(L), Bak, Bax, and method. We successfully grew keratinocytes and fibroblasts from individual newborn mice. Bad, but not Bcl-2, Bcl-W, or Bfl-1. Within the caspase family, mouse keratinocytes express RNA Keratinocytes were maintained in culture for at least six passages without showing any difference for caspases 2, 3, 6, and 8, but not caspases 1, 7, 11, or 12. The only RNA of either family that in cell morphology and growth between affected (ichq/ichq) and normal (ϩ/ϩ or ϩ/ichq). Several showed differential expression in calcium switched cells was Bcl-X(L), which was upregulated. markers of cell proliferation and differentiation have been evaluated by immunofluoresnce staining The upregulation of Bcl-X(L) with switch to high calcium correlates with expression of the protein (Ki 67, keratin 14, calpain I) and other biochemical studies are under investigations. Therefore, in the upper layers of the epidermis. Bcl-X(L) is considered an antiapoptotic protein, but has the generation of cell lines with a known genotype will provide sufficient material to perform recently been shown to be upregulated in certain differentiating hematopoietic cells. We conclude genetic analysis of candidate genes, as well as to establish functional correlates. that Bcl-X(L) may be involved in the terminal differentiation of keratinocytes.

599 600 Loss of Profilaggrin Expression in the Flaky Tail (ft) Mutant Mouse IL-18 and IL-18 Receptor Expression in Human Keratinocytes R. B. Presland,*† D. Boggess,‡ B. A. Sundberg,‡ P. Fleckman† and J. P. Sundberg‡ H. Koizumi, K. Sato-Matsumura, B. Yu, A. Ohkawara and T. Seya Departments of *Oral Biology and †Medicine/Dermatology, University of Washington, Seattle, Department of Dermatology, Hokkaido University School of Medicine, Sapporo; and Department Washington; and ‡The Jackson Laboratory, Bar Harbor, Maine of Immunology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan Flaky tail (ft/ft) is an autosomal recessive mouse mutation that results in stretched and shiny skin IL-18 (IFN-gamma inducing factor) induces IFN-gamma production by Th1 cells and other at birth. At ~5 d to 3 wk of age affected mice show a dry, flaky skin (especially tail and paws) lymphocytes. IL-18 is regarded to play various regulatory reoles in immune system and inflammation and often tail constrictions leading to autoamputation. In previous studies, the ft mutation was of the skin. IL-18 exerts multiple functions through its receptor. Murine keratinocytes have been mapped to mouse Chromosome 3, in the vicinity of a mouse homologue of the epidermal reported to release IL-18, thus we study whether human keratinocytes could be a source of IL- differentiation complex that includes profilaggrin, loricrin, and several S-100 calcium-binding 18 and the presence of IL-18 receptor on keratinocytes. Monoclonal anti IL-18 antibodies and proteins. In this study, we have further characterized these mice by immunohistochemistry and polyclonal anti IL-18 receptor rabbit antibodies were prepared. Formalin embedded tissues and immunoblotting using antibodies to epidermal proteins. Histologically, affected mice had a reduced frozen section of human epidermis, and normal human keratinocytes cultured in serum free modified or absent epidermal granular layer that lacked keratohyalin by EM and showed mild hyperkeratosis. KGM medium were analyzed with immunoperoxidase and immunofluorescence staining. Homo- Immunohistochemistry with a panel of keratin antibodies showed a normal pattern of expression genized cultured keratinocytes were centrifuged, and SDS-PAGE electrophoresis and immunoblot in ft/ft mouse skin. K6 was expressed in hair follicles but not in interfollicular epidermis, consistent were performed. Normal and diseased human epidermis expressed IL-18 in cytoplasm of all living with the relatively mild hyperkeratosis observed. Immunoblot analysis of newborn epidermal epidermal layers, and in some cases of psoriasis IL-18 was in addition strongly expressed in basal extracts showed that profilaggrin/filaggrin was absent from mutant mice; other markers (K14, K1, layer. IL-18 receptor was also expressed in epidermal keratinocytes in cell periphery. With loricrin) were present at comparable levels in control (ϩ/?) and mutant skin. In addition, immunoblot analysis IL-18 receptor was detected only membrane fraction of keratinocytes, not profilaggrin mRNA was present at reduced levels in ft/ft mouse epidermis compared to controls, in cytosol fraction, showing 80 kDa of molecular weight. Our findings document IL-18 and IL- as measured by RT-PCR. These findings suggest that flaky tail is a ‘‘functional’’ profilaggrin null 18 receptor to be present in human epidermal keratinocytes. Keratinocytes do not release IFN- that might underlie the dry, scaly skin characteristic of these mice and may represent a useful gamma. By binding of IL-18 to its receptors on keratinocytes the release of IL-18 might be regulated. mouse model for the human scaling disorder ichthyosis vulgaris where profilaggrin/filaggrin is reduced or absent. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 623

601 602 Analysis of Cytoprotection after Treatments which Induce Heat Shock Proteins in Skin Cells The Similarity Between PV-IgG and Anti-FAS Receptor IgG (FAS-IgG) in Induction of Cultured T. Schmidt-Rose, D. Pollet, G. Sauermann, K-P. Wittern and J. Bergemann. Keratinocytes Apoptosis Paul Gerson Unna-Research Center, Beiersdorf AG, Hamburg, Germany X. Wang, F. Bre´ge´ge`re, M. Frusic-Zlotkin, B. Michel* and Y. Milner In different cellular models elevated levels of heat shock proteins (hsp) have been shown to confer Myers Skin Biochemistry Laboratory, Hebrew University of Jerusalem, Israel; and *Cutaneous increased tolerance against diverse types of stress, including oxidative damage, UV-irradiation and Pathology and Immunofluorescence Lab, AMRIPATH., Beachwood, Ohio chemical agents. In mammals in particular the inducible forms of hsp70 and hsp25 have been We have reported before that keratinocytes in cultures and organ culture respond to PV-IgG linked to cytoprotective effects. It has been suggested, that enhancement of hsp expression prior addition by formation of lesions (‘‘acantholytic’’ lesions) which is the result of induction of to stressful situations might be beneficial for cells. However, few experimental data for skin cells apoptosis. We have shown the accumulation of apoptotic markers (Fas-R, p53, Bax, Fas-L, etc.), exist and knowledge of the molecular details is poor. To elucidate the relevance of hsp-mediated reduction in Bcl-2 and appearance of degraded DNA (TUNEL, agarose gel electrophoresis) in cytoprotection in skin we studied the inducibility of hsp72 by heat and several chemical compounds cultured keratinocytes and human lesional skin, in vivo, induced by PV-IgG. Using immunofluores- in keratinocytes, fibroblasts, and HaCaT cells. Improved experimental conditions allow detection cence techniques (confocal microscopy, image analysis) and immunoblots, we now establish a of even moderate hsp-induction in keratinocytes. We also studied UV-induced DNA-damage and complete similarity between PV-IgG and Fas-IgG induction of lesions and cell death in cultured repair after pretreatments leading to increased expression of heat shock proteins using the comet- keratinocytes as follows: (i) Both IgGs induce the early appearance of soluble (1.5–5 h).and and host-cell-reactivation-assay. Additionally, effects on protein-damage and ubiquitinylation were membranal (5–18 h) Fas-Ligand. (ii) Both IgGs induce the formation of aggregated Fas-R-Fas-L analyzed. We observed significant differences in the iducibility of hsp between the cell types complexes at the cell surface. (iii) Both IgGs recruit caspase 8 (FLICE) and FADD adapter to tested, and favor primary keratinocytes as the more relevant model system for screening substances coaggregate with Fas-R-Fas-L into membranal death particles at the same time scale. (iv) Both to be applied externally to the skin. IgGs activate caspases 1 and 3 (signalling and executioner caspases respectively) in the same kinetics (at 5–18 h). (v) Caspases inhibitor 1 (Ac-YVAD-CHO) inhibited lesions in cultures and recruitment of apoptotic effectors induced by both PV-and Fas-IgG. In summary – PV-IgG is activating the FAS pathway of cell death/apoptosis and via this, apparently, the acantholytic process. Further studies to establish the mode of this activation are under way in our lab.

603 604 Keratin Intermediate Filaments are Stabilized by Conserved Sets of Ionic Charged Residues Membrane Binding Directs Transglutaminase 1 to Specific Glutamines in Involucrin K. Wu, S. Jang, L. Marekov, D. Parry,* J. Yang,† J. Lee‡ and P. Steinert Z. Nemes, L. Marekov and P. Steinert Laboratory of Skin Biology, NIAMS, NIH, Bethesda, Maryland; *Institute of Fundamental Laboratory of Skin Biology, NIAMS, NIH, Bethesda, Maryland Sciences, Massey University, New Zealand; Departments of Dermatology, †Samsung Medical Current theories suggest that an initial step of cornified cell envelope (CE) assembly is the Center, Seoul, and ‡Chungnam National University, Taejon, Korea crosslinking by transglutaminases (TGases) of involucrin directly beneath the plasma membrane. The fundamental building block of keratin intermediate filaments (KIF) is a Type I/Type II These are based largely on data generated from sequencing of peptides obtained from proteolyzed coiled-coil heterodimer molecule. This is stabilized by both hydrophobic interactions of amino fragments of isolated CEs. In order to explore this process further, we describe here an in vitro acid residues in the a and d positions and by ionic interactions of mainly the e and g positions of model system for the crosslinking of involucrin by the TGase 1 enzyme on the surface of the characteristic heptad repeats. Examination of the sequences of all keratin (K) chains has synthetic lipid vesicles (SLV). We used recombinant baculovirus expressed TGase 1, which binds revealed three precisely conserved e-g ionic pairs near the ends of the 1A, 1B and 2B coiled-coil spontaneously to SLV of any composition by virtue of constitutive post-translational fatty acid segments. These are Lys17(g)-Glu22(e) in 1A; Glu84(g)-Lys89(e) in 1B; and Glu106 (g)-Arg111(e) attachments, and recombinant bacterially expressed involucrin. Involucrin binds to SLV of in 2B. There are several other potential pairs of charged residues that are unique to each chain. composition similar to mammalian plasma membranes including Ͼ5% phosphatidylserine, and in To examine the purpose of the conserved sets of ionic pairs, we have studied KIF assembly using a Ca2ϩ-dependent manner. The Ca2ϩ concentration required for involucrin binding is 10-fold bacterially expressed wildtype and mutant K5 and K14 chains in which Glu residues were mutated less than that required for activating TGase 1, which suggests that involucrin binding to the cell to Asp or Ala residues. Mutations of each of the three conserved Glu residues to Asp residues membrane precedes its crosslinking in differentiating keratinocytes. The particulate fraction of resulted in significantly shorter KIF (Ͻ1 µm) as compared to wildtype (Ͼ10 µm), and Ala insect cells expressing TGase 1 incorporated putrescine into only five of 150 Gln residues of substitutions resulted in no KIF. On the other hand, mutations of other nonconserved Glu residues involucrin, of which Gln496 is the most reactive. In contrast, TGase 1 solubilized from insect to either Asp or Ala residues revealed few changes in KIF (2–10 µm). We developed a stability membranes labels 80 of 150 Gln residues. However, when TGase 1 was re-attached to SLV assay in which we measured after crosslinking the urea concentration at which tetramers and carrying involucrin, the same 5 Gln residues were labeled. These data suggest that the keratinocyte dimers could be dissociated. Wildtype tetramers were dissociated by 7 M urea, and dimers by 9 membrane surface acts as a regulatory cofactor ensuring specific steric alignment of both TGase 1 M urea. When either of the conserved Glu residues was replaced by Asp, tetramers were dissociated and involucrin to direct specific crosslinking. We propose a model in which the assembly of the by 5–6 M urea instead, but dimers were unchanged. However, in Ala substitutions, stabilities of CE begins by juxtaposition of TGase 1 and involucrin on keratinocyte membranes followed by both tetramers and dimers were severely compromised. In contrast, Asp or Ala substitutions of consecutive crosslinking as intracellular Ca2ϩ levels rise during terminal differentiation. other nonconserved Glu residues showed only minor changes in stabilities. Together, these data show that: (i) there are three sets of highly conserved pairs of ionic charged residues which are absolutely essential for KIF stability and elongation; (ii) these conserved sets are required for stabilization at the tetramer level of KIF hierarchy; and (iii) other nonconserved charged residue pairs are less critically important in KIF stability.

605 606 Role of CRIK, a Novel Epithelial-Specific Serine-Threonine Kinase, in Keratinocytes Cytokeratin 15 (K15) as an Epithelial Stem Cell Marker: Implications for Aging and Carcinogenesis E. Calautti, S. Cabodi and G. P. Dotto S. Lyle, D. Jih, P. Pyo, Y. Liu and G. Cotsarelis Cutaneous Biology Research Center, MGH and Harvard Medical School, Charlestown, Massa- Department of Dermatology, University of Pennsylvania, Philadelphia, Pennsylvania chusetts We previously identified a monoclonal antibody (DAKO clone C8/144B) that delineates the The small GTPases Rho and Rac play a key role in control of cell structure, motility and adhesion, human hair follicle bulge, and we showed that bulge cells possess properties of hair follicle stem and in linking these changes to control of gene expression and the cell cycle. The cell type- cells. To identify the C8/144B keratinocyte antigen, we screened a human skin cDNA expression specific functions of these GTPases may be explained at least in part by the expression of cell-an library and identified six clones that encoded K15. We confirmed these cloning results by tissue-specific effectors. We have recently identified and isolated a novel serine-threonine kinase, immunoprecipitates of in vitro translated K15 protein with C8/144B. Furthermore, in situ CRIK, which shares significant homology in the kinase domain with previously described Rho hybridization for K15 message resulted in an identical pattern of expression as immunostaining effectors such as ROCK I and II. with the C8/144B antibody. Finally, we raised a polyclonal chicken antibody against a K15- Unlike these latter kinases, CRIK displays an epithelial and brain-specific profile of expression, specific peptide, that immunostained human and mouse hair follicle bulges. We examined K15 suggesting more specialized functions. The kinase can be produced in two isoforms: a ~240 kDa expression in developing and mature skin and determined that K15 is expressed throughout the protein, in which the kinase domain is followed by several functional domains, including a Rho/ basal epidermis and hair follicle bulge during development, but epidermal K15 expression decreased Rac binding site; a ~54 kDa protein (CRIK-SK), which consists mostly of the kinase domain. with age, while the bulge remained intensely positive. These results suggest that the bulge may serve To study the function of CRIK in keratinocytes and its interplay with the Rho/Rac signaling as a repository of epithelial stem cells in adult skin. To test the hypothesis that trichoepitheliomas and pathways, we generated recombinant adenoviruses expressing various mutants of the different basal cell carcinomas arise from hair follicle stem cells, we immunostained a series of these tumors isoforms of the kinase. The effects of CRIK in respect to cell adhesion, cytoskeleton structure, with the K15 antibody. All trichoepitheliomas (13 of 13) and a subset of basal cell carcinomas growth and differentiation of keratinocytes will be described. (BCCs; 10 of 37) expressed K15. Epidermal tumors, including squamous cell carcinomas were K15 negative. Our data demonstate that the stem cell rich bulge areas of human and mouse preferentially express K15. K15 expression in trichoepitheliomas and BCCs suggests that these tumors are derived from hair follicle stem cells in the bulge. 624 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

607 608 Extracellular Matrix Regulates Keratinocyte Plasminogen Activator and Inhibitor Gene Expression Human p120ctn Catenin: Tissue-Specific Expression of Isoforms and Molecular Interactions with J. Bator, R. Cohen and . DA Chambers BP180/type XVII Collagen Center for Molecular Biology of Oral Diseases, Department of Bioengineering, and Department S. Aho, K. Rothenberger and J. Uitto of Biochemistry and Molecular Biology, University of Illinois at Chicago, Chicago, Illinois Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Philadelphia, Plasminogen activators (PAs) and inhibitors (PAIs), mediate plasmin generation and regulate Pennsylvania degradation and remodeling of the extracellular matrix (ECM) during wound healing. Keratinocyte Catenins, a family of structurally related proteins, are involved in epidermal keratinocyte cell-cell migration over the ECM involves integrins that interact with specific matrix components. We adhesion by interacting through their central Armadillo repeats with the intracellular domains of measured increases in keratinocyte PAI-1 mRNA and protein in primary cultures following cadherins, transmembrane components of the adhesion junctions. p120ctn is a catenin expressed in trypsinization and replating on certain substrates. PAI-1 increased in cells plated on vitronectin, different isoforms due to alternative splicing and multiple translation start sites. BP180 is a fibronectin, collagen IV, and the Arg-Gly-Asp (RGD) peptide, as well as on untreated plastic or collagenous transmembrane protein (type XVII collagen) localized to hemidesmosomal attachment nonspecific peptides, suggesting that these ECM proteins may not contribute to PAI-1 upregulation complexes in basal keratinocytes. In this study, we have delineated the molecular interactions in vitro. In contrast, when cells were replated on collagen I, III, or laminin, induction of PAI-1 between these two proteins utilizing the yeast two-hybrid system, which was confirmed by an mRNA and protein was suppressed to control levels. PAI-1 upregulation by soluble factors in vitro protein–protein interaction assay. Specifically, it was shown that an amino-terminal segment including hydrocortisone, epinephrine, EGF, and TGF01β was also suppressed. PAI-1 was not of BP180 (aa. 13–25) contains the information necessary for binding to p120ctn isoforms 1–3, but suppressed by heat-denatured or collagenase-digested collagen, or by antibodies against the collagen not to the isoform 4. This suggested that the interacting domain is located immediately upstream α β α β I-specific integrin 01 201 1. The DGEA peptide, thought to be the 01 201 1-specific sequence from the Armadillo repeats and is encoded by exons 5 and 6, which are subject to alternative in collagen I, was also ineffective in suppressing PAI-1. This suggests that collagen suppression splicing in a minority of transcripts. In addition to epidermal keratinocytes, p120ctn was shown to requires only intact, fibrillar collagen. PAI-1 induction was suppressed by incucbating cells with be expressed in a variety of adult and fetal tissues as well as in a number of human tumors. The genistein, herbimycin A, and cytochalasin D, implicating association with protein tyrosine kinases expression pattern of various p120ctn transcripts, reflecting alternative splicing of the 5Ј exons, was and cytoskeletal changes in the signal transduction pathway. tPA activity and mRNA were strikingly similar between the corresponding adult and fetal tissues, while the expression patterns increased in cells replated on collagen I and III, whereas uPA activity and mRNA levels remain were discordant between certain tumors and their normal parental tissues, suggesting a functional unchanged among all substrates tested These data suggest that expression of keratinocyte PAs and role for the tissue-specific expression of the p120ctn isoforms. Finally, the tissue-specific expression PAIs is differentially regulated by specific matrix proteins, including collagen I, collagen III, of BP180 was shown to partially overlap with that of p120ctn, suggesting that the interaction of and laminin. these two proteins may contribute to the modulation of cell–cell/matrix interactions in such tissues.

609 610 Paraneoplastic Pemphigus Autoantibodies Recognize Epitopes on Plectin that are Shared by Other Psoriatic Keratinocytes Lost Acetylcholine Receptors and its Functions Proteins in the Plakin Family M. Ohtsuyama, C. Matsui, L. Feng and M. Morohashi M. Mahoney, S. Aho, K. Rothenberger, A. Simpson, J. Stanley and J. Uitto Department of Dermatology, Toyama Medical and Pharmaceutical University, Toyama, Japan Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Philadelphia, Acetylcholine (ACh) has been reported to have the effects of differentiation on keratinocytes. On Pennsylvania, and Department of Dermatology, University of Pennsylvania, Philadelphia, the other hand, the effects of ACh on psoriatic keratinocytes, seen in epidermal hyperplasia, are Pennsylvania not well known. We hypothesized that the deficiency of differentiation in psoriatic keratinocytes Paraneoplastic pemphigus (PNP) is an autoimmune blistering skin disease associated with might be due to the loss of sensitivity to ACh. Therefore, we stained ACh receptor immuno- thymoma and lymphoproliferative disorders. We recently showed that PNP patients’ sera contain histochemically and measured intracellular calcium stimulated by ACh because ACh increased autoantibodies against the highly homologous linker region within the carboxy-terminal domain intracellular calcium. Psoriatic tissue was obtained from a nontreated 57-y-old man with psoriasis. of periplakin and envoplakin and that these antibodies recognize common as well as unique The first passage psoriatic keratinocytes were used in this experiment. As a control, normal human epitopes on both proteins. In this study we affinity purified PNP autoantibodies against plectin keratinocytes of neonatal foreskin were purchased from Clonetics Co. Intracellular calcium of and desmoplakin linker regions. These purified antibodies also recognized by immunoblotting and Indo-I loaded cells was observed by laser scanning microscopy. Psoriatic keratinocytes revealed immunoprecipitation other proteins of the plakin family including envoplakin, periplakin, and no change of intracellular calcium concentration and no staining of ACh receptors in psoriatic bullous pemphigoid antigen 1 (BPAG1). Although the PNP whole sera immunoprecipitated an tissue, while normal keratinocytes showed increased intracellular calcium concentration and ACh unidentified 170-kDa protein, purified plectin-and desmoplakin-antibodies did not precipitate this receptors staining. Data suggested that psoriatic keratinocytes lost ACh receptors and its functions protein suggesting that it may not be a member of the plakin family. Computer analysis of the linker region of the members of the plakin family of proteins revealed high sequence homology and similar patterns of antigenic profile, and both shared and unique peaks of antigenicity were detected. Because PNP affects other tissues besides skin, we examined the tissue-specific expression of these plakin transcripts. PCR analysis of multiple tissue cDNA panels revealed that periplakin, plectin and desmoplakin transcripts were present in a variety of fetal and adult tissues. Envoplakin transcript was detected mainly in the kidney, lung and epidermal keratinocytes while BPAG1 transcript was primarily detected in epidermal keratinocytes. These results suggest that plakin proteins may serve multiple functions in a variety of tissues and may play a role in the pathogenesis of PNP.

611 612 Identification of New Keratinocyte Differentiation Candidates Genes by a Genome-Wide Repetin is a Novel ‘‘Fused’’ Protein with Calcium-Binding and Repetitive Domains Localised to Screening Approach Chromosome 1q21 R. Freiberg, C. Barry, P. Brown and P. Khavari M. Huber, G. Siegenthaler VA Palo Alto and Stanford University, Stanford, California 1q21 Consortium* and D. Hohl While specific steps occur in the differentiation of proliferative cells of the epidermal basal layer Dermatology, CHUV-DHURDV, Lausanne and Geneva, Switzerland; *BIOMED2, European into postmitotic suprabasal cells, the gene expression changes responsible for this process are not Union fully understood. To address this issue, we examined gene expression at a genomic scale in normal An important feature of epidermal differentiation is the sequential expression of structural proteins. human keratinocytes in early and later phases of terminal differentiation vs. undifferentiated cells. In order to elucidate the role of human repetin in this multistage process its gene was cloned Cells were cultured in growth media at low confluency [undifferentiated] versus high confluency from a chromosome 1 specific cosmid library using mouse repetin probes. The gene was localised in 1.5 mM calcium media for 6 h [early differentiation] and 48 h [later differentiation]. As to chromosome 1q21 in the region of 100 kbp between the genes of trichohyalin and profilaggrin. confirmed by northern analysis, this treatment strongly induced known epidermal differentiation It has three exons with a translation start site in the second exon. The protein contains 28 genes, such as SPRR I [53-fold] and keratin 10 [129-fold]. mRNA from each cell population glutamine-rich repeats of 12 amino acids (QXXXQGQSSHYG) in the central portion. The was then reverse transcribed, labeled with fluorescent nucleotides or 33P and hybridized to a total sequence of the repeat is very similar to the one of mouse repetin but the number of repeats is of approximately 17,500 genes on cDNA microarrays. Induction of keratinocyte differentiation considerably smaller indicating it is rather the repeat sequence which is functionally important. leads to widespread changes in gene expression, with Ͼ2400 genes induced more than 200% No repeat number polymorphism could be detected in the human population. The N-terminal compared with undifferentiated cell populations. As expected, known epidermal differentiation region showed high homology to the N-terminal domains of profilaggrin and trichohyalin and genes, such stratum corneum chymotryptic enzyme [246-fold], were identified as strongly induced the S100 proteins. This region strongly binds calcium as shown by calcium overlay assays using by this approach. In addition, a total of 548 genes previously uncharacterized in epithelial extracts from bacteria expressing the N-terminal domain of repetin. The C-terminus is highly differentiation were identified for further study, including genes implicated in cell cycle inhibition, conserved between the human and mouse protein indicating its functional importance. Repetin promotion of programmed cell death, transcription and cell adhesion. 58 of these new differenti- mRNA was strongly expressed in methylcellulose suspension cultures but only at very low levels ation-associated genes were ESTs representing genes of unknown function. Of interest, some in submerged high calcium cultures of normal human keratinocytes indicating that repetin is a genes induced by 6 h remained stably expressed at 48 h while others displayed altered expression late differentiation marker. Immunofluorescence analysis suggested that repetin is expressed in the levels between the early and later timepoint, suggesting a gene regulatory pattern reminiscent of upper granular layer of human foreskin and interfollicular skin but not in other epithelial tissues. that seen with early and later T lymphocyte activation/differentiation genes. These data indicate Our findings indicate that repetin belongs to the ‘‘fused’’ subgroup of S100 proteins like profilaggrin that epidermal differentiation impacts expression of a substantial portion of the human genome and trichohyalin. However, differences in expression patterns between repetin, profilaggrin and and identify new candidate genes that may regulate the biologic features characterizing this process. trichohyalin indicate that they have diverse functional roles during epidermal differentiation. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 625

613 614 Caspase Expression and Activity in Human Keratinocytes Topically Applied Lactic Acid Increases Spontaneous Secretion of Vascular Endothelial Growth L. Eckhart,* M. Mildner,* B. Lengauer* and E. Tschachler*† Factor/Vascular Permeability Factor (VEGF/VPF) by Human Reconstructed Epidermis *Department of Dermatology, University of Vienna, Medical School, Vienna, Austria; †CERIES, M. Rendl,* C. Mayer* and E. Tschachler*† Neuilly, France *DIAID, Department of Dermatology, University of Vienna Medical School, Vienna, Austria; Caspases are proteases which mediate the apoptotic degradation of cell components. Since the †CERIES, Neuilly, France various members of this enzyme family represent attractive targets for therapeutic interventions, Alpha-hydroxy acids (AHA) have been widely used for the treatment of photodamage, acne and we have investigated the occurrence and regulation of caspases in keratinocytes. RT-PCR analysis disorders of keratinization. Topical application of AHA results in exfoliation of the outermost revealed that nine of the 10 caspases investigated are expressed in the keratinocyte cell lines layers of the stratum corneum and in stimulation of epidermal turnover. In the present study we HaCaT, A431, in primary keratinocytes cultured in vitro, and in human epidermis. Their were interested whether topically applied lactic acid (LA) could modulate the secretion of cytokines involvement in apoptosis was shown by incubating primary keratinocytes and the cell lines HaCaT by keratinocytes. For this purpose, either creams containing 1.5%, 3%, or 5% of LA or control and A431 with the pan-caspase inhibitor zVAD-fmk. This treatment completely blocked UV-B were topically applied on human epidermal equivalents (EpiDerm) cultured in DMEM. After 24 induced DNA breakdown. Quantification of DNA degradation with an antinucleosome ELISA h supernatants were evaluated for the contents of VEGF/VPF, angiogenin (ANG), and interleukin- and parallel photometrical analysis of caspase 3 activity utilizing a chromogenic substrate revealed 8 (IL-8) by ELISA. At the same time histology of the epidermal samples was obtained. Multiple a high degree of correlation between both parameters after irradiation in the investigated dose apoptotic cells were observed after treatment with 5% LA indicating a toxic effect of LA at this range from 0 to 32 mJ per cm2. To investigate whether de novo transcription contributes to concentration. Treatment with 1.5% and 3% LA increased spontaneous VEGF secretion 2.1- and caspase regulation, we incubated keratinocytes with the transcription inhibitor actinomycin D. 2.5-fold, respectively, as compared to control (mean of three independent experiments performed However, even without additional stimulation, this treatment lead to caspase activation and DNA in triplicate, P Ͻ 0.01). No increase of VEGF/VPF secretion could be detected with 5% LA, fragmentation which also could be blocked by the caspase inhibitor. In agreement with studies which may be due to increased cell death. In contrast to the effects on VEGF/VPF secretion, no on other cell types, our data show that in keratinocytes caspases are indispensible for the apoptotic change of ANG or IL-8 secretion was found with any of the substances tested. In conclusion, response to damaging influences such as UV irradiation and that their activation is independent our data indicate that AHA may exert their effects on skin not only by influencing keratinization of transcriptional activity. but also by modulating the angiostimulatory capacity of keratinocytes.

615 616 Induction of Plasminogen Activator Inhibitor-1 by Hypoxia in Murine Keratinocytes The Role of Mitochondria in Ultraviolet B-Induced Apoptosis of Primary Human Keratinocytes R. Daniel and R. Groves A. Menze, M. Kumar, S. Hurwitz and D. Spandau; Centre for Dermatology, Department of Medicine, University College London, UK Departments of Dermatology and Biochemistry, Indiana University School of Medicine, Indiana- Tissue hypoxia represents a major physiologic change in wounded skin, malignant tumors and polis, Indiana other pathologic skin conditions. Changes in activation of plasminogen, one of the major The response of cells to either pathological or physiological stress induces alterations in mitochondria. proteolytic systems of epidermis, have also been observed in many of these states. One of the It is becoming apparent that these changes in mitochondrial processes are intimately related to the major modulators of plasminogen activation is the plasminogen activation inhibitor-1 (PAI-1) and overall response of the cell to the stress, whether that response is cell survival or cell death. We we were interested to define the effect of hypoxia on production of this inhibitor in the murine examined the role of mitochondria in the response of human keratinocytes to ultraviolet B (UVB) keratinocyte cell line PAM212. PAM212 cells were exposed to normoxic and hypoxic conditions radiation. Cell stress induces the formation of mitochondrial permeability transition (PT) pores at and total RNA was analysed by Northern blot using a PAI-1 specific riboprobe. In resting a contact site between the inner and outer mitochondrial membranes. The formation of keratinocytes low level constitutive expression of PAI-1 was apparent which was markedly induced these mitochondrial PT pores often precedes apoptosis, and the drug bongkrekic acid inhibits following exposure to hypoxic conditions. Time course studies demonstrated induction by 4 h mitochondrial PT pore formation. In order to assess the requirement of mitochondrial PT pore which persisted up to 24 h. To exclude a toxic effect of hypoxia, trypan-blue exclusion studies formation in UVB-induced apoptosis, human keratinocytes were pretreated with bongkrekic acid were performed, and confirmed no loss of keratinocyte viability under the hypoxic conditions up prior to UVB irradiation. No apoptosis was observed in keratinocytes that were pretreated with to 24 h. Hypoxia appeared to have little effect on levels of mRNA encoding urokinase (UPA) or bongkrekic acid and then irradiated with UVB, indicating that the formation of mitochondrial the urokinase receptor (UPA-R). These findings indicate a profound and early sensitivity of PT pores were required for UVB-induced apoptosis. By varying the time at which bongkrekic murine keratinocytes to hypoxia and suggest that tissue oxygen tension may be one stimulus to acid was added to keratinocytes following UVB irradiation, we determined that the formation of the altered proteolytic state of cutaneous wounds. mitochondrial PT pores occurred between four and eight hours post-UVB exposure. A component of the mitochondrial PT pore is believed to be the pro-apoptotic protein BAX. While we did not observe specific changes in the level of BAX expression following UVB irradiation, we did detect a change in BAX-related protein interactions following UVB exposure that correlated with the induction of apoptosis. We conclude that UVB-induced apoptosis of human keratinocytes involves the formation of mitochondrial PT pores and the association of these pores with BAX.

617 618 Identification of an S100A2 Binding Protein in Human Keratinocytes Long-Term Culture of Epidermal Keratinocytes from Single Newborn Mice T. Woods, S. Stoll and J. Elder B. Hager, J. Bickenbach and P. Fleckman Department of Dermatology, University of Michigan, Ann Arbor, Michigan Department of Medicine (Dermatology), University of Washington, Seattle, Washington; Depart- S100A2 was identified on the basis of decreased expression in tumor-derived cell lines relative to ment of Anatomy and Cell Biology, Univ of Iowa, Iowa City, Iowa their normal counterparts. Based on patterns of overexpression in tumors, wounded skin, and As more transgenic and null mice are produced with skin abnormalities, the ability to establish psoriasis, we have postulated a role for S100A2 in regenerative epidermal differentiation. ErbB cultures of mouse epidermal keratinocytes has become increasingly important. This requires that activation appears to play a central role in this process; we have shown that transcription of each cell line be derived from a single mouse. Since the number of epidermal keratinocytes from S100A2 is strongly induced by EGF in normal human KC (NHK). Based on structural homologies a mouse is limited and often insufficient for adequate analysis, such cultures require multiple to calmodulin, we hypothesized that S100A2 exerts its function by binding to one or more passages. Freezing the cells would allow keratinocytes from several mouse lines to be compared target proteins. A study was therefore undertaken to identify S100A2 binding partners by at one time. However, mouse keratinocytes have been exceedingly difficult to grow as primary coimmunoprecipitation, using a specific mouse anti-S100A2 mAb (J Cell Sci 110:611, 1997). cultures, and subculturing these cells has been virtually impossible until now. We describe a NHK were seeded at 5000 cells per cm2 in complete KGM. At 60%–70% confluence, the cells method that allows the growth and subculture of total mouse keratinocytes from single newborn were labeled with [35S]-methionine for 4 h and lysed in low-stringency lysis buffer (25 mM Tris pups for at least 19 subcultures, allowing the increase in cell number by seven logs. Epidermal 8.0, 140 mM NaCl, 14 mM KCl, 0.1% NP-40) or RIPA (10 mM Tris 7.4, 150 mM NaCl, 1% keratinocytes from newborn mice were grown on selected lots of collagen IV coated dishes in NP-40, 1% deoxycholate, 0.1% SDS), each containing proteinase inhibitors. IPs utilized either murine fibroblast conditioned medium with 0.06 mM calcium and added growth factors. The anti-S100 A2 mAb or MOPC21 isotype control (30 mg per ml). In either lysis buffer, IP of cells grew to confluence, could be passaged, frozen as viable stocks, and induced to differentiate. S100A2 was strongly reduced by excess EGTA, suggesting that S100A2 undergoes Caϩϩ- Biochemically and morphologically the cultured keratinocytes demonstrated a pattern characteristic dependent conformation changes. Both buffers yielded several coimmunoprecip-itated bands. of basal cells. Stratified cultures which made mouse keratin 1 and profilaggrin were induced However, only RIPA buffer yielded bands not seen in the isotype control. Using RIPA, we through passage 10 by purging the monolayer cultures of growth factors, then adding medium consistently identified a strong band of Mr 01µ 125 kDa in NHK derived from three different with 0.15 mM to 0.5 mM calcium. This method should be of use to investigators with interest donors. Interestingly, this band was equally abundant in A431 cells and NHK, but expressed only in mouse keratinocyte biology and to those who are now trying to analyze skin phenotypes of at low levels in HaCaT cells. We and others have previously shown that S100A2 forms a transgenic or knockout mice. homodimer, and numerous other S100-interacting proteins have been identified by in vitro reconstitution experiments. However, with the exception of p11-annexin II binding, these experiments are the first to demonstrate the interaction of an S100 protein with an abundant target protein in intact cells. 626 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

619 620 Laminin 5 Expression in Acute and Chronic Wounds 01β-Adrenergic Receptor Activation Inhibits Keratinocyte Migration by a cAMP-Independent M. Fujita,* M. L. Usui,* W. G. Carter,†‡ M. C. Ryan*‡ and J. E. Olerud* Pathway Departments of *Medicine (Dermatology) and †Pathobiology, University of Washington, Seattle, J. Chen,*† E. Z. Jong,* B. B. Hoffman† and R. R. Isseroff* Washington; ‡Fred Hutchinson Cancer Research Center, Seattle, Washington *Department of Dermatology, UC Davis School of Medicine, California; and †Department of Chronic, nonhealing foot and leg ulcers are the most common cause of lower extremity amputation Medicine, Stanford University School of Medicine, Stanford, California in patients with diabetes mellitus. Keratinocytes (KC) in the epidermal tongue of tissue from Keratinocyte migration plays an important role in re-epithelialization and skin wound healing. ulcers of patients with diabetes proliferate extensively yet do not appear to migrate across the Although growth factor receptors have been well studied in the process, little is known about the chronic ulcer bed. Migration of KC, essential to wound healing, depends on normal integrin– role of G protein-coupled receptors (GPCR). There is increasing evidence that GPCRs cross- receptor interactions. Laminin 5 interacts with the 01α301β1 integrin receptor to mediate talk with growth factor receptor-mediated signal transduction in a variety of cell types. We therefore attachment during KC migration and with the 01α601β4 integrin receptor to mediate the hypothesize that GPCRs are involved in growth factor-induced migration in keratinocytes. In subsequent stable adhesion. We were interested in evaluating laminin 5 in the chronic ulcers of this study, we have addressed the effects of 01β-adrenergic receptor (01β-AR), a Gs protein- patients with diabetes and in acute, incisional wounds of 1–28 d duration of age-matched normal coupled receptor, on cell migration and signaling mechanisms in cultured human keratinocytes. volunteers. We used immunohistochemical methods to evaluate tissue for the presence of the By using computer-based image analysis of cell migration, we have demonstrated that isoproterenol, 01α3 integrin (P1F2), the 01α3 chain of laminin 5 (C2–5), a precursor form of the 01α3 chain a01β-AR agonist, significantly reduces cell migration stimulated by EGF, extracellular Ca2ϩ,or of laminin 5 (D2–1) thought to be secreted as an early event of KC migration and the proliferation HKGS (Human Keratinocyte Growth Supplement) in a dose-dependent manner, with maximal marker Ki67. In situ hybridization using a 600-bp digoxygenin labeled probe for the 01α3 chain of 80% inhibition. The inhibition is completely reversed by pretreatment of the cells with the of laminin 5 was used to determine expression of laminin 5 mRNA. Staining for the 01α3 integrin 01β-AR antagonist, timolol, suggesting that isoproterenol is acting on specific 01β-adrenergic (P1F2), the 01α3 chain of laminin 5 (C2–5) and Ki67 was present on the ulcer margin from receptors. 01β-AR activation also induces changes in the actin microfilament network, characterized patients with diabetes and on incisional wounds at all time points. Staining for the precursor form by a disorganization of F-actin fibers and increased vinculin-positive focal adhesions. To determine of the 01α3 chain of laminin 5 (D2–1) was present on the 1, 3 and 7 d acute wounds, but not if these effects are mediated through Gs-protein activation of adenylate cyclase (cAMP signaling on the 14 and 28 d acute wounds. Surprisingly, D2–1 staining was not present on the ulcer pathway), cells were pretreated with an adenylate cyclase inhibitor, 2’5’-Dideoxyadenosine (DDA). margins of the patients with diabetes. Laminin 5 mRNA was expressed in the 1, 3 and 7 d acute DDA pretreatment significantly inhibits isoproterenol-induced cAMP production in keratinocytes wounds, but not in the 14 and 28 d acute wounds. Laminin 5 mRNA was not expressed in the but does not reverse isoproterenol-induced inhibition of migration. These findings suggest that chronic ulcers. The absence of the precursor form of the 01α3 chain of laminin 5 on the 01β-AR-mediated inhibition of keratinocyte migration is independent of the classic cAMP proliferative KC at the diabetic ulcer margin and lack of laminin 5 mRNA expression suggests signaling pathway, and may offer new insights into therapeutic approaches to wound healing. that the precursor form is not being actively synthesized by those cells. We speculate that the precursor form of laminin 5 may be important for KC migration. Characterization of the laminin 5 isoforms that maximize cell migration could provide important tools for wound healing therapy.

621 622 Downstream Targets of Urokinase Plasminogen Activator-Mediated Signal Transduction in Augmented Anti-Tumor Immunity by Immunization with Epidermal Cells Infected with an Human Keratinocytes Adenovirus Vector Containing a cDNA for GM-CSF E. Jong, J. Chen and R. Isseroff H. Ozawa, K. Seiffert, N. Hackett, N. Topf, R. G. Crystal and R. D. Granstein Department of Dermatology, University of California, Davis School of Medicine, Davis, California Department of Dermatol. and Div. of Pulmonary and Critical Care Medicine, Weill Medical The urokinase plasminogen activator (uPA) and its receptor (uPAR) is thought to play an important College of Cornell University, New York, New York role in keratinocyte migration and wound healing. Recently the uPA/uPAR mediated intracellular GM-CSF is an important maturation factor for epidermal Langerhans cells and exposure to GM- signaling pathways have received more attention and, depending on the cell type studied, different CSF is necessary for Langerhans cells to be able to present tumor-associated antigens (TAA) for kinase pathways have been implicated. In this study we investigated the downstream kinase induction of substantial antitumor immunity when used to prime naive mice. To evaluate the pathways activated by uPA. In cultured, low passage, human keratinocytes starved of growth ability of adenovirus vectors (AdV) to transfer and express genes to epidermal cells (EC), EC were factors and serum, addition of uPA (2–10 nM) results in transient activation of p38 MAP kinase infected with a vector containing a cDNA for green fluorescence protein. Green fluorescence was (MAPK). Activation, evidenced by phosphorylation, was maximal at five minutes, returning to detected in ~30% of keratinocytes by FACS analysis. To examine whether introduction of a basal levels at 10 min. The extracellular signal regulated kinase 1 (ERK 1 MAPK) is also activated cDNA for GM-CSF into EC would enhance their ability to present TAA, we infected EC from a/d transiently in response to uPA stimulation with a similar time course. However, uPA does not CAF1 mice (H-2 ) with an AdV containing a cDNA insert for murine GM-CSF (AdGM-CSF). activate ERK 2 MAPK. Since the F-actin polymerization modulator, heat shock protein 27 (HSP Control cells were infected with a null vector (AdNull). EC infected with AdGM-CSF produced 27) is a known downstream target of the p38 MAPK, we investigated whether uPA treatment of a much greater amount of GM-CSF (by ELISA) than EC infected with AdNull. When cultured keratinocytes results in phosphorylation of this protein. Increased levels of phosphorylated isoforms with allogeneic T cells, AdGM-CSF-infected EC had significantly greater allostimulatory capacity of HSP 27 were observed after uPA treatment of cells, and this phosphorylation was partially than AdNull-infected EC [35,616 Ϯ 11,935 (SD) CPM for EC/AdGM-CSF vs 21,175 Ϯ 4664 abrogated by the specific p38 inhibitor, SB 203580. This is the first demonstration of the signaling for EC/AdNull (P Ͻ 0.03)]. One day after infection, both cell types were pulsed with a soluble pathway of uPA in keratinocytes. These findings suggest that the p38 MAPK pathway signals for extract of the S1509a spindle cell tumor (H-2a) as a source of TAA, washed three times, and 5– 5 uPA, and results in HSP 27 activation and possibly actin rearrangement. By modulating p38 10 01ϫ 10 cells injected s.c. into each of five CAF1 mice. Control mice were immunized with MAPK activity, uPA may thus contribute to cell motility. cells not pulsed with TAA. Primings were repeated weekly 01ϫ 2 and 7 d after the last priming each mouse was challenged s.c. with 2 01ϫ 106 living S1509a cells. Tumor growth was scored over time. Tumors in mice immunized with EC/AdGM-CSF pulsed with TAA were significantly smaller than in all other groups [at day 18-648 mm3 for EC/AdGM-CSF ϩ TAA: vs 2294 mm3 for EC/AdNull ϩ TAA (P Ͻ 0.02), vs 2093 mm3 for EC/AdGM-CSF-TAA (P Ͻ 0.002), vs 1796 mm3 for EC/AdNull-TAA (P ϭ 0.001)]. Infection of EC with an AdV containing GM- CSF cDNA significantly augments their ability to present TAA for induction of in vivo antitumor immunity.

623 624 Failure of Fas Mediated Activation Induced Cell Death in Se´zary Cells Derived from a Patient HIV-Infected Human Epidermal Langerhans Cells Migrate into and Infect Human Lymphoid Tissue with Se´zary Syndrome A. Blauvelt, S. Glushakova and L. Margolis S. Meech, K. Ricketts, P. Walsh, R. Duke and . DA Norris Dermatology Branch, NCI and NICHD, Bethesda, Maryland Univ of Colorado HSC, Denver, Colorado During sexual transmission of HIV, transport of virus from mucosal surface dendritic cells, i.e. Mycosis fungoides (MF) and Se´zary Syndrome (SS) are types of cutaneous T cell lymphomas that Langerhans cells, to CD4ϩ T cells within regional lymph node is postulated, although there is involve the massive accumulation of skin associated mature, CD4ϩ activated memory T cells. little experimental evidence to support this theory. In this study, we describe an ex vivo model to We propose that MF and SS result from a disorder of apoptosis rather than from a proliferative study Langerhans cell migration and HIV transmission to human lymphoid tissue. Langerhans cells advantage. To limit clonal expansion, activated lymphocytes are programmed to self-destruct via were isolated from normal-appearing skin (which resemble Langerhans cells within mucosal apoptosis. Congenital defects in Fas or Fas ligand are associated with the failure of activation epithelium) of healthy volunteers, allowed to mature in culture for 24 h, and then exposed to induced cell death (AICD) and the subsequent development of lymphoproliferative syndromes. various dilutions of a panel of primary and laboratory HIV-1 isolates for 4 h. HIV-infected The purpose of this study was to determine if Se´zary cells (SC) fail to undergo Fas mediated Langerhans cells were then washed extensively to remove excess virus and applied to surfaces of AICD. After ficoll separation, an isolate containing Ͼ99% SC was obtained from a patient with cultured tonsil tissue blocks (obtained from routine tonsillectomies performed on HIV-seronegative SS. These cells expressed markers of activated, memory cells. Memory cells normally express Fas, individuals). To determine tissue levels of HIV infection, HIV p24 protein was measured in however, Ͻ20% of these SC cells expressed Fas. Fas expression was induced to near normal levels culture media surrounding tonsils by ELISA. All primary and laboratory HIV isolates could be by mitogens and growth factors (e.g. ConA, IL-2, IFN-alpha, and anti-CD3 antibody). Unlike transmitted by Langerhans cells and establish high levels of infection in lymphoid tissues (p24 Ͼ normal controls, the Fas expressed on these Se´zary cells failed to induce substantial cell death 10 ng per ml). Interestingly, depletion of Langerhans cells within epidermal cell suspensions upon cross-linking by its physiologic ligand in a chromium release killing assay (Ͻ35% Se´zary cell abrogated the ability of HIV-exposed suspensions to transmit virus to tonsils. In addition, dye- lysis compared to Ͼ85% cell lysis in normal controls). We are able to restore Fas mediated AICD labeled Langerhans cells could be seen within tissue sections within hours after applying Langerhans in these deficient Se´zary cells via biochemical approaches. cells to tonsil surfaces. Thus, this experimental system relies on the migratory abilities of HIV- We have shown that a profound defect in Fas mediated AICD exists in a patient with Se´zary infected Langerhans cells to traffic into lymphoid tissue and transmit infection. In summary, we syndrome. Such a defect may explain why Se´zary cells, which do not appear to have a proliferative have developed a novel ex vivo model for human Langerhans cells trafficking into lymphoid tissue, advantage, nonetheless accumulate over time in vivo. and have shown that Langerhans cells, purported initial target cells for HIV, are responsible for transmitting virus to T cells using this system. Importantly, this model should prove useful in further understanding very early events of primary HIV infection and in testing particular agents designed to block this process. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 627

625 626 The Chemokine Receptor Expression Profile and Chemokine Responsiveness of Human Dendritic Langerhans Cell Development From CD14ϩ Hemopoietic Precursors Cells is Dependent on Cell Subtype and Maturational Stage S. Jaksits, E. Kriehuber, G. Stingl and D. Maurer A. Charbonnier, A. Rot, G. Stingl and D. Maurer DIAID, Department of Dermatology, University of Vienna Medical School, Vienna, Austria DIAID, Department of Dermatology, University of Vienna Medical School; Novartis Research A major breakthrough in the understanding of Langerhans cell development came from the Institute, Vienna, Austria observations that the exposure of CD34ϩ hemopoietic stem cells (SC) to GM-CSF/TNF-01α Chemokines (CK), via triggering of specific cell surface-bound receptors, can execute dendritic gives rise to a progeny of CD1aϩ, E-cadherin (E-cad)ϩ, Birbeck granule (BG)-containing cells cell (DC) migration and, thus, may decisively control the proper tissue localization of these cells with immunostimmulatory properties strikingly resembling those of Langerhans cells isolated from in vivo. Using GM-CSF/TNF-01α/Flt3-L-elicited human hemopoietic stem cell cultures, we human skin. In vitro, GM-CSF/TNF-01α-mediated Langerhans cell development occurs via a analyzed the CK-R expression profiles of CD1aϩCD14– Langerhans cell and CD1a–CD14ϩ non- CD1aϩ/CD14– intermediate stage. Besides this CD1aϩ Langerhans cell precursor, CD14ϩ/CD1a– Langerhans cells DC precursors along their differentiation into the respective mature DC subsets cells emerge early during the culture and, if isolated and further propagated in the presence of in vitro. At day 6 of culture, both the Langerhans cells and the non-Langerhans cell DC precursors GM-CSF/TNF-01α and M-CSF, develop into CD1aϩ/FXIIIaϩ but E-cad–/BG– dendritic cells express a broad array of CC- and CXC-CK-R (i.e. CCR-1, Ϫ2, Ϫ3, Ϫ5, Ϫ6; CXCR-2 and (non- Langerhans cell DC) and CD14ϩ/CD1a– monocytes, respectively. In addition to GM- Ϫ4). While non-Langerhans cell DC maintain this broad CK-R expression pattern of their CSF/TNF-01α, transforming growth factor-01β1 (TGF-01β1) can be of importance for Langerhans precursor stage until day 12, Langerhans cells continue to express CCR6 and CXCR4 but loose cell development as evidenced by the findings that TGF-01β1–/– mice harbor a major defect in β all the other CK-R-encoding mRNAs. The MIP-301 /ELC-specific receptor CCR7 was found Langerhans cell development and that TGF-01β1 amplifies in vitro Langerhans cell development to be the only CK-R species that is de novo expressed selectively during the late stages (i.e. after from CD34ϩ SC. However, the nature of the TGF-01β1-responsive Langerhans cell progenitor day 12) of Langerhans cells and non-Langerhans cell DC development in vitro. To see whether remains unknown. In an attempt to identify and characterize this precursor, we isolated CD1aϩ/ this differentiation/maturation-related CK-R expression profile of in vitro generated Langerhans CD14– and CD14ϩ/CD1a– progenitor cells at day 6 of the culture and propagated both cell cells is compatible with the in vivo situation, human epidermal Langerhans cells were isolated and populations in the presence or absence of TGF-01β1 until day 12–14. Interestingly, the most analyzed both immediately after their purification and following ex vivo maturation. In their pronounced TGF-01β1-mediated Langerhans cell-promoting effect was observed in the CD14ϩ/ immature state, epidermal Langerhans cells express transcripts encoding CCR-2, Ϫ6, Ϫ7 and – β CXCR-4. During their ex vivo maturation they loose CCR-2 and Ϫ6 transcripts while maintaining CD1a progenitor cell population. In the presence of TGF-01 1, a major portion of these cells CCR-7 and CXCR-4 expression. The comparative analysis of the CK-R profiles of in vitro acquired not only CD1a but also E-cad, displayed the BG-associated lag antigen and contained typical BG by electron microscopy and, thus by (immuno)phenotype, was essentially indistinguish- generated and ex vivo isolated Langerhans cells suggests that, within the normal human epidermis, ϩ – Langerhans cells occur at different maturational stages. Accordingly, the most recently immigrated able from the Langerhans cell population emerging from CD1a /CD14 progenitors. Importantly, epidermal Langerhans cells population should still express CCR-2 and Ϫ6 whereas the most the latter cell population gave rise to a Langerhans cell progeny whether or not the cultures were mature Langerhans cells which are in progress of leaving the epidermis should already express supplemented with TGF-01β1 or neutralizing anti-TGF-01β Abs. To see whether this cytokine ϩ CCR7. The biologic importance of this maturation-related CK-R expression pattern was further exerts a similar Langerhans cell-promoting effect also on more mature members of the CD14 analyzed by chemotaxis assays. As expected by their broad CK-R repertoire, day 6 Langerhans cell lineage, i.e. monocytes/monocyte-derived (md)DC, we comparatively analyzed the immuno- cells/DC progenitors displayed chemotactic responsiveness to a broad panel of CC-(MIP-101α, phenotypes of TGF-01β1-exposed mdDC and CD14ϩ/CD1a– progenitor-derived Langerhans RANTES, MIP-301α) and CXC-CK (IL-8, SDF-101α). Surprisingly, MIP-301α preferentially cells. While TGF-01β1 could induce E-cad in both cell populations, certain antigens which are attracted CD1aϩ Langerhans cells rather than CD14ϩ non- Langerhans cell progenitors. In normally absent from Langerhans cells, i.e, FXIIIa and CD11b, occurred in TGF-01β1-exposed contrast, SDF-101α induced migratory responses of poorly differentiated CD1a–CD14– cells but mdDC but were not expressed by LC derived from CD14ϩ/CD1a– progenitor cells in the hardly attracted single-positive progenitors. While the E-cadherin (E-cad)ϩ progeny of CD1aϩ presence of TGF-01β1. These data show that TGF-01β1 exerts its Langerhans cells-promoting Langerhans cell precursors lost its MIP-301α responsiveness until day 12, around day 14, both E- effect at the level of a CD14ϩ/CD1a– progenitor and, furthermore, suggest that the in vivo cadϩ Langerhans cells and E-cad– non- Langerhans cell DC start to display pronounced migratory relevant Langerhans cell precursor could be a CD14ϩ rather than a CD1aϩ cell homing to and/ responses to MIP-301β. Thus, it appears that Langerhans cell/DC progenitors due to their wide or residing in the skin. CK-R expression profile may be able to enter different tissues in response to various CC-or CXC-CK species. Our results further suggest that the most likely CK candidate involved in attracting Langerhans cells into the epidermis is the CCR-6 ligand MIP-301α whereas the CCR- 7 ligand MIP-301β may be involved in the emigration of various skin DC populations and/or in their proper localization within T cell areas of skin-draining lymph nodes. 627 628 Green Fluorescent Protein Expressed in Murine Dendritic Cells Normal Skin and Cutaneous T-Cell Infiltrates Often Harbor Clonally Restricted B Cells – B. E. Rich, L. Liu* and T. S. Kupper Implications for the Molecular Biological Diagnosis of Cutaneous B-Cell Lymphomas (CBCLs) Harvard Skin Disease Research Center and *Center for Neurological Diseases, Brigham and M. Nihal, D. Mikkola and G. Wood Women’s Hospital, Boston, Massachusetts Department of Dermatology, Skin Diseases Research Center, Case Western Reserve University Recent findings have pointed to the pivotal role of dendritic cells in the uptake of antigen and and the VA Medical Center, Cleveland, Ohio presentation to T cells. In an effort to further our understanding of the biology of dendritic cells A monoclonal B-cell population is the hallmark of CBCL. PCR-based assays involving amplification we have generated transgenic mice in which transcriptional control elements of the human CD11c of immunoglobulin heavy chain (IgH) gene rearrangements give rise to one or two dominant gene direct expression of a convenient marker, modified jellyfish green fluorescent protein (GFP), bands depending on whether one or both IgH alleles are rearranged in the tumor clone. We have to dendritic cells. The integrin CD11c (also known as p150,95) is principally expressed by dendritic tested a variety of IgH PCR assays and determined that the most reproducible are a nested cells and the murine CD11c promoter region has previously been used to direct expression of multiplex method employing variable (V) region primers complementary to framework (FR)1 transgenes to dendritic cells. In the present study, a cassette vector suitable for generating transgenic and FR2 domains, and a non-nested method employing V region primer complementary to the constructs was assembled containing approximately 5 kb of the 5Ј region of the human CD11c FR3 domain. Using these assays, we have analyzed a variety of cutaneous samples including gene (generous gift of John Noti, Guthrie Research Institute, Sayre, PA, Noti et al, DNA and normal skin, chronic dermatitis, mycosis fungoides, cutaneous lymphoid hyperplasia and CBCLs, Cell Biology, 1992, 11:123–138) and noncoding SV40 sequences that contain an intron and as well as polyclonal and monoclonal controls including normal blood, reactive tonsils and B-cell transcriptional termination/polyadenylation signals. We have inserted a cDNA encoding GFP lymphoma cell lines. As expected, the IgH PCR assays produced diffuse smears (agarose gels) or (EGFP, Clontech) into this vector and used the resulting construct to generate transgenic mice by complex ladders (polyacrylamide gels) in the blood and tonsil polyclonal controls, and one or two pronuclear injection. Dendritic cells from these CD11c-GFP mice accumulate GFP in their distinct bands in the monoclonal B-cell line controls. However, in normal skin and many non- cytoplasm and are inherently fluorescent. Therefore these cells can be detected by flow cytometry CBCL lesional skin samples, one or a small number of discrete bands were detected. In many and fluorescent microscopy without any manipulation. The expression pattern of this transgene cases, this made it impossible to distinguish true positives (monoclonal CBCL) from false positives parallels that of the endogenous CD11c gene in blood and spleen cells as well as in cultured bone (clonally restricted benign B cells). Correlation with immunophenotyping results confirmed that marrow cells. This model provides a powerful tool for the study of dendritic cell biology. We are false positive results were confined to samples with sparse B cell populations. These findings continuing to further characterize these mice and to utilize them for studies of dendritic cell biology. suggest that the molecular diagnosis of CBCL using IgH PCR assays should be reserved for B- cell-rich infiltrates, and may be unreliable for the detection of so-called ‘‘T-cell-rich CBCLs’’ such as cutaneous lymphomatoid granulomatosis. Nevertheless, they also document the presence of clonally restricted B cells in normal skin that may represent the B-cell component of the ‘‘skin- associated lymphoid tissue’’ (SALT), and that may be the source of B cells in cutaneous lymphoid hyperplasia and CBCLs. 629 630 Consistently High Sensitivity of PCR/DGGE Analysis for the Detection of Dominant Clonality Differential Tyrosine Kinase Display of Benign and Malignant T Cells in Cutaneous T-Cell Lymphomas (CTCLs) J. Siddiqui, D. Robinson, H-J. Kung and G. Wood D. Mikkola, N. Horvath, A. Gilliani, S. Stevens, T. Spiro, K. D. Cooper and G. Wood Departments of Dermatology and Molecular Biology/Microbiology, and the Skin Diseases Departments of Dermatology, Medicine and the Skin Diseases Research Center, Case Western Research Center, Case Western Reserve University and the VA Medical Center, Cleveland, Ohio Reserve University and the VA Medical Center, Cleveland, Ohio Tyrosine kinases (TKs) play crucial roles in regulating cell activation, proliferation, differentiation The PCR/DGGE technique involves the amplification of T-cell receptor (TCR)-y chain gene and apoptosis. Because of their strong relationship to growth, there is a disproportionately high rearrangements (GR) followed by separation of the genomic PCR products using denaturing chance for them to become oncogenes. At least one third of known oncogenes are TKs. TK gradient gel electrophoresis (Wood et al. J Invest Dermatol, 103:34, 1994). The assay uses DNA abnormalities have been documented in a wide variety of neoplasms, and correlate with both extracted from fresh-frozen tissue, and has a sensitivity of about 1% when a T-cell clone is diluted malignant transformation and tumor progression. As the signal pathways of various TKs are in polyclonal T cells. We use variable (V) region primers that detect Vy 1–9 gene segments but becoming clarified, cross-talk among different ones is emerging as the rule rather than the not Vy 10 or 1 lNe use joining (J) region primers that detect Jy 1 and 2 gene segments but not exception. Therefore, it is likely that an aggregate TK profile will serve as a better tumor marker JYP, P1 or P2. Because the TCR-y gene has two alleles that are often clonally rearranged in than any single TK. A novel method has been developed recently that allows the determination CTCLs, only one GR needs to be detectable in order to document dominant tumor clonality. In of the entire TK profile of a cell population (Robinson et al. PNAS 93:5958, 1996). This approach our 1994 series, we found 61 of 68 cases of mycosis flingoides/Sezary syndrome (MF/SS) exhibited involves: (i) RT/PCR of TK transcripts using a single set of degenerate primers complementary dominant clonality by PCR/DGGE. Since then, we used PCR/DGGE to analyze an additional to homologous regions shared by all TKs; (ii) digestion of RT/PCR products with a selected set 100 cases of unequivocal CTCLs. Dominant clonality was detected in 80 cases (80% true positives) of restriction endonucleases; (iii) identification of individual TKs via comparison of restriction while it was not seen in 20 cases (20% false negatives). The true positive cases included 68 MF, fragments to a defined database. Using this technology, we have identified several putative TK ϩ three SS, five CD30 CTCLs, three CD30-CTCLs and one adult T-cell lymphoma. The 20 differences between normal peripheral blood lymphoid cells and neoplastic T cells such as HUT- false negative cases were all MF, including many early cases with only patch lesions. Potential 78. Once differences are identifed, they can be confirmed by nucleotide sequencing and/or RT/ explanations for false negative results include TCR GR that are gemiline, deleted or involve segments not detectable with the primer combinations used. In addition, a turnor clone density PCR assays specific for individual TKs. This analysis should allow us to define differences in TK below the detection threshold of the assay may occur in early skin lesions with only sparse expression that may be involved in the pathogenesis and/or progression of cutaneous T- lymphoid infiltrates. Although the combined true positive rate of our 1994 and current series (141 cell lymphomas. of 168 or 84%) is favorable compared to other published PCR assays, our findings suggest the possibility that additional PCR primer combinations may improve the sensitivity of PCR/DGGE analysis and decrease the proportion of false negative results. These are being tested. 628 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

631 632 Intradermal GM-CSF Alters Cutaneous Antigen Presenting Cells and Differentially Effects Local Imiquimod, a Topical Immune Response Modifier, Induces Migration of Langerhans Cells Versus Distant Immunization in Humans H. Suzuki, B. Wang, G. M. Shivji, P. Toto,* P. Amerio,* M. A. Tomai,† D. McDermott,† R. S. Stevens,* I. Kremer, J. DiCarlo and K. Cooper* Miller† and D. N. Sauder Case Western Reserve University, University Hospitals of Cleveland, and *VA Medical Service, Division of Dermatology, Sunnybrook, Health Science Centre, University of Toronto, Toronto, Cleveland, Ohio Canada; *Department of Dermatology, University ‘‘G.d’Annunzio’’, Chieti, Italy; †3M Pharma- Granulocyte-Macrophage Colony stimulating factor (GM-CSF) is a pleiotropic cytokine which ceuticals, St. Paul, Minnesota augments dendritic cell growth and antigen presenting cell (APC) function. We hypothesized that Langerhans cells are bone marrow derived dendritic cells that represent the major antigen presenting intradermal delivery of GM-CSF would alter the number and differentiative state of APCs and cells in the skin. Langerhans cells take up and process antigen within the epidermis and present immunization strength at the injection site, but not at a distant site. GM-CSF (0–250 mg), or processed antigen to T lymphocyte in the regional lymph nodes and thus form an integral part of placebo, were administered to intradermal buttock skin daily for four days. Biopsy specimens were the cutaneous immune response. The cutaneous immune response can be modified by a number then obtained and stained for S-100, CD1a, CD40, CD68, HLA-DR, and ICAM-1. In the of pharmacological and physical agents. For the most part these agents act by suppressing immune epidermis, CD1aϩ S-100ϩ Dendritic Cells (DC) were reduced in number and were rounder with function. Recently, a topical immune response modifier, imiquimod has been shown to enhance shortened dendrites. In the papillary dermis, DC were more numerous, particularly perivascularly. the cutaneous immune response. Imiquimod has antiviral and antitumor effects in animal models In the deep dermis, CD68ϩ macrophages were increased in number, without predilection for and the biological activity of imiquimod in part is due to its effect as a cytokine inducer. perivascular localization. Expression of the APC activation markers HLA-DR, CD40, and ICAM- Preliminary data suggested that imiquimod could effect on Langerhans cells. In order to clarify 1 was increased throughout the skin and colocalized to areas of increased APC numbers. The 125 this effect on Langerhans cells, we examined Langerhans cell morphology and migration in mg dose of GM-CSF optimally induced DC and activation markers without inducing adverse imiquimod treated skin. After topical imiquimod treatment, changes in the density and morphology effects and was used for immunization studies. Subjects were sensitized to DNCB through GM- of Langerhans cells were observed. The density of Iaϩ cells decreased 2 d after treatment, falling CSF (or placebo-) treated skin and to DPCP through untreated skin at a distant site. GM-CSF to approximately 43% by day 10. The Ia positive in cells remaining in the skin appeared larger treated sites were significantly more erythematous. Subjects immunized through GM-CSF treated and more dendritic suggesting an activated state. ATPase staining of epidermal sheet confirmed sites exhibited 64% greater elicitation responses (skin edema) to DNCB than did placebo-treated the Ia obtaining findings. Two-color flow cytometric analysis of the lymph node cells showed subjects. GM-CSF treated subjects showed 43% lower responses to DPCP than did placebo- increased Iaϩ cell population suggesting that imiquimod enhanced Langerhans cell migration from treated subjects. The difference between DNCB (local) and DPCP (distant) responses was skin to draining lymph nodes. This enhanced Langerhans cell migration was also associated with significantly greater for GM-CSF treated subjects than for placebo responses (n ϭ 8, P Ͻ 0.05). an enhanced contact hypersensitivity. These results suggest that the mechanism of modulation of Intradermal GM-CSF alters local macrophagic and dendritic APC numbers, distribution and immune response by imiquimod is in part due to effects on Langerhans cells. activation state. Differences in immune responses at the site of GM-CSF administration and at distant sites have ramifications for microbial or tumor vaccine protocols which utilize GM-CSF to augment immunization efficiency.

633 634 Deletion Mutagenesis Reveals Majority of Soluble Human SCF Protein Required for KIT p53-Dependent Chemosensitivity in Melanoma Cells Receptor Binding/Activation G. Li, J. Bush and V. Ho J. Obadiah, J. Burch and J. Grichnik Department of Medicine, Division of Dermatology, University of British Columbia, Vancouver, Departments of Medicine and Cell Biology, Duke University Medical Center, Durham, North BC, Canada Carolina Metastatic melanomas are often resistant to chemotherapy. To study whether the p53 status affects Stem cell factor (SCF) appears to have the capacity to regulate homeostasis of cutaneous melanocytes chemo sensitivity, we compared the responses to chemotherapy of four melanoma cell lines which through activation of its receptor, KIT. As a membrane bound protein, SCF is 248 or 220 residues contain wild-type p53 and four cell lines which carry mutant p53. We found that the melanoma in length. The larger protein can be enzymatically cleaved to release an active glycosylated 165- cells with wild-type p53 had significantly higher response to the treatment with cisplatin, residue fragment. Non-glycosylated recombinant proteins including 164 of the N-terminal residues camptothecin, and vincristine. To further confirm the role of p53 in chemo sensitivity in melanoma of SCF are capable of activating melanocytes. Our interest was to determine whether significantly cells, we transfected a mutant p53 expression vector, pEDI, into melanoma cell line MMAN smaller recombinant fragments of the human SCF protein could be identified that would function which contains wild-type p53. Introduction of mutant p53 into MMAN cells resulted in faster as either agonists or antagonists of KIT activation. Therefore, we constructed a series of 5 N-and growth rate, rendered the cells more resistant to chemotherapy, and inhibited anticancer drug- 5 C-terminal deletion mutants of SCF linked to GST (N-terminal) to allow for ready purification. induced apoptosis. The bel-2 expression was decreased after treatment with camptothecin in the We established a CELISA based system utilizing a melanoma line and a cell flow assay utilizing a parental MMAN cells, while bcl-2 expression was increased in cells overexpressing mutant p53. leukemia line to follow the loss of KIT antigen with exposure to SCF. While the parent GST– Conversely, the expression of bax as decreased in cells overexpressing mutant p53. To confirm SCF molecule demonstrated KIT down-regulation, deletion of 27 N- or 50 C-terminal SCF that bax:bcl-2 ratio is important for chemo sensitivity in melanoma cells, we transfected a bax residues prevented this activity in both systems. Since it was possible that the some of the truncated expression vector into MMAN cells. Transient overexpression of bax enhanced camptothecinind- SCF proteins would bind but not activate KIT (possible competitive antagonists), a third assay uced apoptosis. Taken together, our results indicate that expression of wild-type p53 may enhance system was established utilizing an ELISA based system to quantitate the binding of soluble KIT chemosensitivity in melanoma cells by regulating bax and bcl-2 expression. protein. This assay revealed that deletion of 27 N-terminal or 50 C-terminal residues also resulted in loss of KIT binding. These findings suggest that the majority of the recombinant GST-SCF protein is required for KIT binding. This full length fusion protein will be useful in the study of melanocytes and KIT; however, it appears that significantly smaller fragments of this fusion protein will not act as agonists or antagonists without further modification.

635 636 Calcitonin Gene-Related Peptide Upregulates Melanogenesis and Enhances Melanocyte Dendricity Role of Keratinocyte-Derived Cytokines in Hypopigmentation in Vitiligo Vulgaris Via Induction of Keratinocyte-Derived Melanotrophic Factors R. Kitamura, K. Tsukamoto, S. Shimada and G. Imokawa M. Toyoda, T. Makino, C. Matui, L. Yu and M. Morohashi Department of Dermatology, Yamanashi Medical University, Yamanashi; and Kao Biological Department of Dermatology, Toyama Medical and Pharmaceutical University, Toyama, Japan Science Laboratories, Tochigi, Japan It has recently been shown that cutaneous axon terminals and epidermal melanocytes make contact The pathogenesis of vitiligo vulgaris (VV) is generally documented to be involved in the following via chemical synapses in human skin and that calcitonin gene-related peptide (CGRP) induces three hypothesis: (i) the autoimmune disease, (ii) the neural abnormality, and (iii) the autocytotoxic melanocyte proliferation. To further clarify the effect of neuropeptides on the biology and phenomena of melanocytes. However, the precise mechanisms still remain to be clarified. Recent morphology of melanocytes, especially with respect to melanogenesis and melanocyte dendricity, studies have demonstrated the important roles of keratinocyte-derived cytokines in the regulation organ cultures of normal human skin and cultured melanocytes were exposed to various of melanocyte functions in a paracrine fashion. Therefore, we hypothesized that changes in the neuropeptides present in intraepidermal nerve endings. Of the neuropeptides examined, skin epidermal circumstances including secretion of regulatory cytokines may cause melanocytes to exposed to CGRP in organ culture showed increases in melanocyte number, epidermal melanin decrease their melanogenic function or to be unable to stay in the epidermis. To determine content, melanosome number and degree of melanization. CGRP alone had no significant effect whether the potential of epidermal cells to produce several melanogenic cytokines is altered in on melanogenesis of cultured melanocytes, whereas the addition of medium conditioned by the lesional epidermis, we have compared using immunohistochemistry and reverse transcription CGRP-stimulated keratinocytes (CGRP-KCM) induced melanogenesis as indicated by biochemical polymerase chain reaction (RT-PCR), the expression of SCF, ET-1, bFGF, GM-CSF and HGF assays of tyrosinase activity and melanin content. Furthermore, CGRP-KCM significantly enhanced between nonlesional and lesional epidermis. Immunohistochemical analysis in the lesional epidermis melanocyte dendricity, a crucial factor affecting epidermal pigmentation. These findings suggest revealed a weak immunostaining with antibodies to SCF and ET-1 as compared with a definite that keratinocytes produce and secrete some melanotrophic factors following stimulation with staining in the perilesional normal epidermis. RT-PCR of these transcripts demonstrated that CGRP, which modulate growth, melanin synthesis and dendricity of melanocytes. These data there is an accentuated expression of SCF, ET-1 and GM-CSF transcripts in the lesional epidermis demonstrate intimate interactions between the cutaneous nervous system and melanocytes within over the nonlesional epidermis. These findings suggest that the deterioration of the ET-1 and SCF the epidermal environment. productions within keratinocytes at post-transcriptional levels may be associated with the dysfunction of melanocytes in VV. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 629

637 638 The Role of the Hermansky-Pudlak Gene Product in Intracellular Trafficking of Melanogenic Independent Regulation of Growth and SMAD-Mediated Transcription by TGF-01β in Human Proteins Melanoma Cells R. E. Boissy and Y. Zhao A. Mauviel, T. Nishiyama and U. Rodeck Department of Dermatology, University of Cincinnati College of Medicine, Cincinnati, Ohio Thomas Jefferson University, Philadelphia, Pennsylvania To understand the inherent basis for the reduced pigmentation in the Hermansky–Pudlak Syndrome Increased production of TGF-01β coupled with resistance to the growth inhibitory effects of (HPS), cultures of melanocytes were developed using skin biopsies obtained from patients lacking TGF-01β is characteristic of several types of neoplasia including human melanoma. In select the expression of HPS mRNA. Characteristic ultrastructural features of the HPS melanocytes epithelial malignancies, lack of TGF-01β induced growth inhibition is associated with disruptions consist of: (i) large membrane bounded complexes containing membranous chambers, unpigmented of TGF-01β-dependent SMAD signaling and transcription. Using a recently developped SMAD- and pigmented melanosomes, irregular deposits of DOPA reaction products, and granular/ responsive reporter construct in transient cell transfection experiments, we demonstrate intact amorphous material sometimes resembling the cytoplasm; and (ii) DOPA-positive rings delineated SMAD-dependent transcription in human melanoma cells regardless of their proliferative response on both sides by limiting membranes, and (iii) DOPA positive 50 nm vesicles throughout the to exogenous TGF-01β. Furthermore, in some melanoma cell lines constitutive SMAD-dependent cytosol. These structures suggest that tyrosinase was aberrantly translocated in the HPS melanocyte. transcription was observed which was due in part to endogenous TGF-01β. These results establish The expression of tyrosinase related protein-1 (TRP-1) and granulophysin, exhibited a large that resistance of melanoma cells to TGF-01β-induced growth inhibition occurs independently granular pattern of expression throughout the HPS melanocytes, that appears to correlate with of intact TGF-01β receptor/SMAD-mediated transcriptional regulation. They also suggest that the large membrane complexes observed ultrastructurally. Antibodies to a synthetic portion of the melanoma-derived TGF-01β may exert autocrine effects on SMAD-sensitive target genes. HPS protein (i.e. aa 253–267) were generated and recognized a 80–83 kDa protein in normal melanocytes absent from HPS melanocytes. Indirect immunofluorescent cytochemistry using the HPS antiserum with confocal microscopy demonstrated that the HPS gene product was prominent in the cell body appearing predominantly perinuclear with a cisternal network-like pattern resembling endoplasmic reticulum localization and conspicuously reduced in the Golgi zone. In addition, a fine granular staining pattern was distributed throughout the cytosol and dendrites indicating melanosomal localization. These observations are consistent with the hypothesis that the HPS gene product is involved in organellogenesis. We propose that the HPS gene product regulates recognition/incorporation of Golgi-derived vesicles containing melanocyte specific proteins with preformed premelanosomes.

639 640 New Results with Melanoderm, an Epidermal Model Containing Functional Melanocytes Detection of Melanoma Cells by Tyrosinase and TRP-1 Sequences in the Peripheral Blood Using M. Klausner, P. Neal, B. Breyfogle and J. Kubilus Reverse Transcriptase and Polymerase Chain Reaction MatTek Corporation, Ashland, Massachusetts H-Y. Jin, T. Yamashita and K. Jimbow We have previously reported on the successful incorporation of normal human melanocytes Department of Dermatology, Sapporo Medical University School of Medicine, Sapporo, Japan (NHM) into a highly differentiated, three-dimensional tissue culture model of human epidermis Malignant melanoma is one of the highly metastatic cancers among all the human malignancies. (J Invest Dermatol, 104:616, 1995). Melanocytes exhibited dendritic morphology, were localized The early detection of metastatic lesions, followed by surgical excision, however, improves the in the basal cell layer, and converted l-dopa to melanin. Previously, spontaneous pigmentation of overall prognosis of melanoma patients and even may cure them. Currently the serum and/or the melanocytes could not be observed using light microscopy except in the presence of l-dopa. urinary excretion of melanin precursors, e.g. cysteinyl dopa and 6H5 M12C, have been used for However, recent improvements in the culture medium formulation now yield cultures in which detection of latent melanoma metastases. Tyrosinase and its related protein (TRP) are exclusively melanin containing NHM can be observed as early as 10 d after seeding. Over a 4-wk period, expressed by melanocytes and melanoma cells. To develop a new tool for identification of latent cultures become increasingly pigmented with retention of normal epithelial morphology. Cultures metastatic melanoma, we have developed a molecular assay using nested reverse transcriptase containing NHM derived from black donors show increased pigmentation versus those containing polymerase chain reaction (RT-PCR), which amplifies tyrosinase and TRP cDNAs, and examined caucasian derived NHM; both types of cultures were distinctly darker than NHM-free cultures. which one of tyrosinase gene family will be useful for detection of circulating melanoma cells. These results suggest that this model will be useful to study melanogenesis, skin pigmentation, We found that TRP-2 mRNA was not useful for DNA diagnosis of melanoma cells, since it was and other photobiological effects on skin in vitro. amplified from healthy donor cells. In contrast, tyrosinase and TRP-1 mRNAs were not detectable in any of the healthy donors or patients with other malignancies. Our nested PCR amplifies 180 base-paired (bp) fragment located in the 3rd exon for tyrosinase and 204 bp fragment in the 2nd exon for TRP-1. In the reconstruction experiment, the nested PCR was able to detect two to five melanoma cells in 6 ml of normal blood samples. When plasmids containing tyrosinase and TRP-1 cDNAs were used for the sensitivity assay, 0.2 ag tyrosinase and 0.2 pg TRP1 plasmid per 50 l of reaction buffer could be detected by the agarose gel electrophoresis. Our approach of the detection of combined tyrosinase and TRP-1 mRNA sequences, not tyrosinase mRNA alone as having studied recently by several other investigators, should provide a new tool for early detection of melanoma patients with potentially high risks of metastasis.

641 642 Characterisation of a Melanocyte-Derived RT-PCR Inhibitor Increased Epidermal Inducible Nitric Oxide Synthase in Vitiligo J. Bach,* L. Eckhart* and E. Tschachler*† A-K. Kaczorowska, L. Nelson and K. Schallreuter *Department of Dermatology, University of Vienna, Medical School, Vienna, Austria; †CERIES, Clinical and Experimental Dermatology, Department of Biomedical Sciences, University of Neuilly, France Bradford, UK Because of its high sensitivity, reverse transcription-polymerase chain reaction is a powerful method A wide variety of biological effects exerted by nitric oxide (NO), makes this signalling molecule for the investigation of gene expression in cell types from which it is difficult to prepare large an important mediator in both physiological and pathological states. As all skin cell types express amounts of RNA such as melanocytes. nitric oxide synthase (NOS) constitutively, it is apparent that the activity of NO is required for When we reverse-transcribed RNA from normal human epidermal melanocytes grown in vitro maintaining skin homeostasis, regulating processes of vasodilatation, melanogenesis and also cell and performed PCR analysis of several transcripts, we noticed that considerably weaker RT-PCR differentiation and apoptosis. The expression of inducible nitric oxide synthase (iNOS) in skin has signals were obtained than from comparable amounts of RNA prepared from other cell types. In been shown in several dermatological disorders such as psoriasis, neoplasia and allergy. This order to test the hypothesis that an inhibitor was present, we added aliquots of melanocyte cDNA prompted us to investigate the presence of iNOS in full skin biopsies obtained from involved and preparations as well as melanocyte RNA preparations to other cDNA or plasmid DNA templates. uninvolved areas and in melanocyte cultures established from uninvolved epidermis of patients Indeed, PCRs were inhibited in a concentration-dependent manner. Since both the melanocyte with vitiligo. Immunohistochemical staining employing monoclonal antibodies specific for iNOS lysates and the final cDNA preparations were colored brown, we investigated melanin as a showed a significant increase of enzyme expression in the upper layers of epidermis in involved candidate inhibitor. The water-soluble fraction of synthetic melanin was found to be a potent skin from patients with vitiligo. Staining was negative in healthy controls. Melanocytes established inhibitor of Taq polymerase, whereas reverse transcriptase was blocked only at more than tenfold from nonlesional skin demonstrated a significant increase of iNOS compared to control melanocytes. higher melanin concentrations. PCR inhibition could be overcome by diluting the template Our findings are in agreement with the evidence for overproduction of (6R) L-erythro 5,6,7,8 DNA-melanin mix or by using higher enzyme concentrations, which suggests that direct binding tetrahydrobiopterin, an essential cofactor required for the activity of NOS, previously reported in of melanin to Taq polymerase is involved. Additionally, size exclusion chromatography using a patients with vitiligo. We would like to suggest that the high levels of iNOS found in vitiliginous sephadex G100 column was effective in the removal of the inhibitor from mixtures of synthetic skin could contribute to oxidative stress, which has been implicated in the pathogenesis of vitiligo. melanin with template DNA and from melanocyte-derived cDNA preparations. These findings A wide variety of biological effects exerted by NO, makes this signalling molecule an important suggest that it would be advisable to check RT-PCRs from melanocyte RNAs for the presence mediator playing a significant role in both physiological and pathological states. As all cell types of the inhibitor and to apply the discussed countermeasures in the case of such a contamination residing in skin express nitric oxide synthases (NOS) constitutievely, it is apparent the activity of in order to avoid false-negative results. NO is required for maintaing skin homeostatsis, regulating proceses of vasodilation, melanogenesis and also presumably cell differentiation and apaptosis. The expression of inducible nitric synthases (iNOS) in skin has been shown in several varoius dermatoses: skin tumours, allergy and psoriasis. This prompted us to investgate the presence of iNOS in biopsies obtained from lesional and nonlesional vitiliginous skin. and melanocyte culture established from nonlesional epidermis a patient with vitiligo. Immunohistochemial staining of full skin biopsies of vtiligo patients showed a significant increase of iNOS expression in the upper layers of epidermis. No staining was found in healthy controls. Staining of melanocytes established from vitiliginous nonlesional skin showed a significant increase of iNOS presence compared to control melanocytes. Our findings are in line with the evidence of overproduction of 6-tetrahydrobiopterin, as essential coffactor required for the activity of NOS. We suggest that high level of iNOOS found in vitligous skin may contribute to the oxidative stress in skin reported previously in vitiligo. 630 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

643 644 Molecular Diagnosis of Metastatic Skin Disease Using Laser Capture Microdissection Quantification of Lateral Thermal Effect of CO2 and Erbium-YAG Lasers S. Milchgrub, I. Witsuba, B. Kim, C. Rutherford, J. Urban, A. Gazdar and P. Cruz Jr C. R. Shea, N. S. Sadick, and V. G. Prieto Departments of Dermatology, Internal Medicine and Pathology, UT South-western Medical Department of Pathology and Medicine (Dermatology), Duke University Medical Center, Durham, Center, Dallas, Texas North Carolina, and New York Hospital-Cornell Medical Center, New York, New York A 57-y-old woman had erythematous telangiectatic plaques with scarring alopecia involving the The CO2 laser has been used as a substitute for the cold steel knife to create a receptacle for skin scalp, and palpable cervical lymph nodes (LN). Four years prior, she had duodenal bleeding due punches during hair transplantation. By setting the laser to a determined intensity, the depth of to a carcinoid that was resected. CBC, liver enzymes, chest Xray, abdominal CT scan and urine the resulting defect can be made relatively constant. It has been suggested that the addition of an 5-HIAA were normal. Histology of skin and cervical LN showed aggregated foamy cells, whereas Erbium-YAG laser may result in decreased hemorrhage of the skin defect, by thermally damaging that of archival duodenal tumor showed pseudoglandular polygonal cells. Immunohistochemistry the blood vessels and inducing coagulation. However, this addition may cause unacceptable showed duodenal tumor reactivity to chromogranin, synaptophysin and somastostatin, consistent damage to the surrounding tissue. This study investigates possible interactions of this combination ϩ with carcinoid. By contrast, scalp and LN tumors were (–) for these markers, but ( ) for CD68, of laser irradiations. antitrypsin and lysozyme. Lack of consensus diagnosis due to histologic and immunophenotypic Scalp specimens obtained from transplant-recipient areas of patients with androgenetic alopecia disparities led us to use laser capture microdissection (LCM) to procure carcinoid cells from the duodenal tumor and clear cells from scalp and LN tumors. Lymphocytes were also microdissected were irradiated with Erb-YAG laser (1.7 J) alone or in combination with CO2 laser (5 or 10 W). as source of constitutional DNA. DNA was extracted and PCR performed to evaluate loss of As a control, a 1-mm punch was used in adjacent skin to produce a standard surgical defect. The heterozygosity (LOH) and microsatellite alterations (MA) using primers flanking 22 micro repeat defects were then removed along with adjacent skin using a 3-mm punch and processed for polymorphisms from nine chromosomal regions including genes for MEN1 (11q), CDKN2 (9p), routine histology. As an additional control, a 3-mm punch biopsy was taken from untreated p53 (17p), and bronchial carcinoid (3p). Fourteen of 22 markers were informative (heterozygous adjacent skin. All specimens were stained with hematoxylin and eosin, von Gieson (elastic tissue), in control lymphocytes). Marker D3S1274 on 3p12 showed LOH of the same parental allele in Masson trichrome (collagen and muscle), and PAS (glycogen and mucopolysaccharides). The the three tumors. D3S4103 on 3p14.2 showed identical shifted bands indicative of the same MA extent of thermal damage was recorded as a range between the minimum, maximum, and average in the three tumors. These abnormalities indicated a common clonal origin, leading to a diagnosis diameter of altered collagen or epithelium around the defect. of 1° duodenal carcinoid with metastasis to scalp and cervical LN, and treatment with radio- and The thermal damage from Efb-YAG was similar to that from Erb-YAGϩ CO2 at SW (range: immuno-therapy (rather than chemotherapy). Our experience represents a novel and valuable 0.02–0.18 mm). However, when CO2 was added at 10 W, there was a greater zone of thermal application of LCM and PCR analysis of genomic markers to identify the origin of metastatic disease. coagulation (range: 0.05–0.50 mm). As expected, minimal lateral damage was detected in specimens incised with cold steel knife. These data indicate that the addition of CO2 to Erb-YAG laser causes enhanced thermal damage in a dose-dependent manner. When employed at 5 W it may permit a better hemostasis without significantly increased thermal damage to surrounding structures. 645 646 PTEN Protein Expression in Normal Skin and in Basal Cell Carcinoma Does Confocal Reflectance Microscopy Help Demarcation and Screening of Non-Pigmented H. Zhang, J. T. Celebi, X. Ping, H. Tsou and M. Peacocke Skin Neoplasms? Department of Dermatology, Columbia University, New York, New York S. Gonza´lez,* M. Rajadhyaksha,*† G. Menaker,† J. L. Fewkes‡ and R. R. Anderson* Cowden syndrome (CS) is an autosomal dominant inherited condition, associated with germline *Wellman Laboratories of Photomedicine, †Den-natology Surgery, Department of Dermatology, PTEN mutations, and characterized by benign and malignant tumors in specific organ systems, Massachusetts General Hospital, Harvard Medical School, ‡Mohs/Dermatologic Surgery, Mass particularly the skin, the breast and the thyroid. Although breast and thyroid cancers are prevalent Eye and Ear Infirmary, Boston, Massachusetts; §Lucid, Henrietta, New York in individuals with this disease, the presence of basal cell carcinomas have also been reported. Near infrared confocal reflectance microscopy (CM) provides noninvasive real-time images of Functional in vitro studies indicate that PTEN, a putative tumor suppressor gene, is a dual specific thin en-face skin sections with high resolution and contrast. The lateral resolution is 0.5–1.0 µm phosphatase. To further characterize the PTEN protein expression in normal skin and in basal cell and axial resolution (‘‘section thickness’’) is 3–5 µm. Imaging of cells, nuclei, other organelles, carcinomas (BCC), we performed immunohistochemical staining with PTEN antibody and Ki67, microvessels, and hair follicles can be done at resolution comparable to standard histology, up to a marker of cellular proliferation. Initially, polyclonal antiserum to the PTEN recombinant protein a maximum depth of 250–300 µm. We have already characterized nonpigmented skin malignances was generated in a series of rabbits, and the antisera was affinity purified. Western blotting using (BCC, SCC) based on their unstained, native histologic features using CM. Live dysplastic both the preimmune and the immune serum demonstrated the recognition of a 47-kDa band, the keratinocytes with nonanaplastic patterns are viewed in case of BCC, and variable degree of size of the predicted PTEN protein, by the immune but not the preimmune serum. Then, paraffin anaplasia and individual keratinization figures within the epithelium in SCC. A prominent embedded sections of normal human skin and BCCs were stained by using the affinity purified vasculature together with a dermal inflammatory infiltrate was seen in some of the cases. Real- PTEN antibody. Of the 10 cases analyzed, all showed strong PTEN staining of the spinous layer time confocal imaging has shown that we can demarcate lateral microscopic margins in vivo based of the epidermis and the inner root sheath of the hair follicle, whereas Ki67 showed no staining on architectural markers at dermo–epidermal level. In vivo CM provides an unprecedented of these areas, but strong expression at the basal cell layer and the outer root sheath of the hair histologic view of living skin. In addition, the use of CM for ex vivo screening of freshly excised follicle. While the examination of the superficial and nodular type BCCs revealed decreased skin specimens show that detection of neoplastic tissue and infiltrating cells without destroying staining with PTEN, an increased expression of Ki67 was noted. These data suggest that, PTEN, with its tumor suppressor function, is expressed in the more differentiated cells of the epidermis the specimen is possible. This communication will outline challenges for real-time screening and the hair follicle, but not in the proliferating cells. process of excised tissue during Mohs’ surgery. Our data shows that potential applications of CM may include precise real-time detection and screening of neoplasms both in vivo and ex vivo which may eventually lead to confocal imaging-guided surgeries including biopsies and Mohs’ micrographic surgery. 647 648 New Insights into the Applicability of T Cell Receptor 01γ Gene Rearrangement Analysis in Primary Cutaneous B-Cell Lymphomas are Descended From Germinal Center B Cells a Cutaneous T Cell Lymphoma Molecularbiological Characterization on the Single Cell Level N. Li and J. Bhawan S. Rutz, S. Gellrich, P. Lorenz, C. Jacobs, S. Golembowski, M. v. Zimmermann, S. Lippert, H. Dermatopathology Section, Department of Dermatology, Boston University School of Medicine Audring, W. Sterry and S. Jahn Boston, Massachusetts Department of Dermatology, Medical Faculty (Charite´), Humboldt University Berlin, Germany γ Detection of clonal T cell receptor 01 gene rearrangement (T-rearrangement) by PCR and B cell lymphomas are designated as primary cutaneous when occurring in the skin with no denaturing gradient gel electrophoresis is a marker for cutaneous T cell lymphoma (CTCL). To evidence for systemic involvement at the time of diagnosis. Since the pathogenesis of these determine the accuracy of histologic criteria alone for the diagnosis of CTCL, 74 patients with lymphomas is poorly understood, our study aimed at elucidating the origin of the malignant B- cutaneous T cell infiltrates were first reviewed by two independent dermatopathologists and their cells, mechanisms of their clonal evolution, and local distribution within the skin lesion. confidence in the diagnosis of CTCL was assigned one of four levels. Then the specimens were To address these questions from a molecularbiological point of view, single B lymphocytes were analyzed for T-rearrangement. The following results were obtained: mobilized from tissue cryo-sections and their immunoglobulin gene rearrangements were analyzed Histologic criteria Number of cases Positive clonal % error using a multiplex single cell PCR approach which permits the simultaneous amplification of the T cell rearrangement Ig heavy and light chain gene rearrangement from one cell. Eight patients with primary cutaneous B-cell lymphomas were studied. From each patient a sufficient number of clonal rearrangements could be amplified. Ig gene analysis of 549 cells Diagnostic 10 10 0 revealed a situation comparable to MALT-type lymphomas: (i) different variable region genes are Consistent 11 8 27 used; (ii) an antigen-driven selection and expansion is indicated by the observed pattern of somatic Suggestive 27 15 44 hypermutation; (iii) the occurrence of intraclonal diversity (ongoing mutation) implies a germinal- nondiagnostic 26 3 89 center cell origin of the malignant B cells; (iv) the proportion of clonally related B cells in the whole B cell infiltrate may vary significantly in patients. Furthermore, we did not observe any differences regarding the Ig genes when comparing the two clinical subentities of cutaneous B- In six patients two or three biopsies were analysed for T-cell rearrangement: cell lymphomas: lymphoma of head and trunk, and lymphoma of the lower extremities. In our experiments, we could demonstrate the necessity to analyze different biopsies of the same patient Case Histologic criteria Clonal T cell rearrangement when applying a single cell approach in order to reliably detect intraclonal diversity and thus to (1st/2nd/3rd biopsy) (1st/2nd biopsy) deduce the clonal evolution. The obtained Ig gene rearrangements are the starting point for the expression of Fab fragments in E. coli to identify the specificity of the antibody produced by the malignant cells which may help to further elucidate the pathogenesis of primary cutaneous B- 1 Diagnostic/consistent Yes/yes cell lymphomas. 2 Consistent/suggestive Yes/yes 3 Consistent/consistent Yes/yes 4 Suggestive/suggestive Yes/yes 5 Suggestive/nondiagnostic Yes/No 6 Suggestive/nondiagnostic/suggestive No/yes/no

In eight cases T-rearrangement was compared between formalin fixed and fresh specimens of the same individual, but with different degrees of histologic confidence (no lower than suggestive). In all cases fresh specimens were positive. In five of the cases (2-diagnostic, 3-suggestive) formalin fixed specimens were positive as well, and in three cases (1-consistent, 2-suggestive) formalin fixed specimens were negative. Our data demonstrate that T-rearrangement studies are consistently positive regardless of tissue fixation when the histologic degree of confidence is very high (diagnostic). However, T- rearrangement studies are particularly important in earlier cases with less conclusive histology. In these cases, multiple biopsies may be required to establish the diagnosis and analysis of fresh tissue increases the sensitivity. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 631

649 650 Clonally Expanded T Lymphocytes in Mycosis Fungoides – Detected by Micromanipulation and Scanning EM in 32 Cases of Suspicious Onychomycosis Single-Cell PCR Lei Pengcheng, Jian Huahui and Dong Lihui S. Gellrich, A. Lukowsky, S. Rutz, J. M. Muche, S. Jahn, H. Audring and W. Sterry Department of Dermatology, 3rd Hospital, Beijing Medical University, Beijing, China Department of Dermatology, Medical Faculty (Charite´), Humboldt-University Berlin, Germany Thirty-two cases of suspicious onychomysosis were repeated examined by mycologic examination According to the EORTC classification of primary cutaneous lymphomas, Mycosis fungoides (microscopic and culture), the result were negative. Scanning EM, histologic examination and (MF) represents an epidermotropic T-cell lymphoma. Molecularbiological and histological data mallclipping-grinding-powder culture were processed. suggest that the portion of clonal T cells among the total cellular infiltrate may vary extensively Twenty-six cases (81.2%) were positive in scanning EM. Twenty-two cases (68.75%) were between different patients and even between different stages of the disease. positive in histologic examination. Twelve cases (37.5%) were positive in nail-clippirig-grinding- Until recently, the precise localization of neoplastic T cells remained unclear (intraepidermally, powder culture. within the so called Pautrier’s microabscesses, within the subepidermal infiltrate). The application EM showing how the fungus destroying and penetrating the nailplate on the nailventral, developing of micromanipulation and single-cell PCR for amplifying the TCRg chain gene rearrangements and destroying the nail on the horizontal plane and damaging nail on the surface plane. is an optimal tool to address this question and was used for the first time for studying cutaneous Onychomycosis is an increasing fungal infection of the nail, microscopy and culture are the T cell lymphomas (CTCL). diagnostic are ‘‘golden standard’’. The problem is the pseudonegtive and pseudpositive. High All together, 176 CD4 positive T lymphocytes were isolated from three patients with MF. From suspicious onychomycosis, which were repeated negative in mycologic examination, are distressed 57 T cells, a PCR product was obtained. A number of 38 of these products could be sequenced dermatologist frequently. (efficiency 22%). From one patient, 36 epidermotropic T cells were analyzed. In this way, 14 The three new examination, especially scanning EM, help to raise the rate of diagnosis, and clonal and three other sequences were detected. Interestingly, the clonally related T lymphocytes give us a picture to know how the fungus invade the nail, and can help us to know the drug harbor a biallelic rearrangement. In five of these T cells, rearrangements of both alleles could be how to kill the fungus. amplified whereas in another four cells only one allele was found. T lymphocytes from the band- like infiltrates were taken from all three patients. However, no identical TCR01γ chain rearrangements were observed in this fraction. In summary, clonally related T lymphocytes were detected exclusively within the population of epidermotropic cells, but not in the subepidermal band-like infiltrate. This indicates, that the neoplastic T cells are located within the epidermotropic fraction of the skin infiltrating T lymphocytes.

651 652 The ‘‘Second Wave’’ of Hair Growth in Hairless Mouse Skin: S Model to Study Mechanisms of Perinatal Development of Murine Skin Innervation Anagen Hair Follicle Induction E. M. J. Peters, V. A. Botchkarev,* D. J. Tobin, S. Mueller-Roever* and R. Paus* A. A. Panteleyev,* R. Paus,‡ J. P. Sundberg§ and A. M. Christiano*† Department of Dermatology, Charite´, Humbold Universita¨t, Berlin, Germany; *Department of *Department of Dermatology and †Genetics and Development, Columbia University, New York, Biomedical Sciences, University of Bradford, Bradford, UK New York; ‡Dept of Dermatology, Charite´, Humboldt-University, Berlin, Germany; §The Jackson Morphogenesis of skin and epithelial appendages may require complex neuro-epithelial and neuro– Laboratory, Bar Harbor, Maine mesenchymal interactions. Here, we used immunoreactivity (IR) to several neuronal and blood The understanding of the as yet elusive molecular and cellular mechanisms that initiate hair follicle vessel-related structural proteins, enzymes, and peptides to study the pattern and timing of back (HF) growth as a result of epithelial–mesenchymal interactions during early anagen is one of the skin and hair follicle (HF) innervation in C57BL/6 mice. In addition, the location of perifollicular major goals of hair biology. The hairless phenotype in hr/hr mutant mice results in complete loss nerve fibers (NFs) was confirmed by electronmicroscopy. Both approaches allowed us to investigate of HF integrity and subsequent separation of its functional portions such as the dermal papilla the development of skin NFs in relation to HF and blood vessel development from day 14 (DP) and putative bulge cells, thus providing an attractive model for dissecting the interactions estimated gestational age (E14) to the postnatal day 1 (P1, day of birth). First PGP 9.5-IR NFs between different HF cell populations. Our histological and immunohistological studies of HRS/ appeared in the dermis as early as E 15 before any evidence of HF morphogenesis was detected J hr/hr mouse skin (days 19–70 pp) revealed that during the final stage of HF disintegration (days morphologically. At E16, stage 1/2 tylotrich HFs were present and were associated with large 19–20 pp), the few epithelial cells that remain associated with the detached DP remnants become caliber NFs [PGP 9.5-, GAP 43-, choline-acetyltransferase (ChAT)-IR], which gave rise to fine Ki-67ϩ, suggesting a high proliferative activity. This fibroblast-associated keratinocyte proliferation NFs that extended towards the epidermis and to the interface of the epidermis and the emerging results in the formation of functional anagen HFs around week 5 pp. However, these HFs are HFs. These HF-associated NFs later form the follicular (neural) network A (FNA). Conspiciously, disoriented and largely unable to reach the skin surface. These findings suggest that isolated DP developing NFs run parralel to the developing vasculature of the skin, but only a few fibers were cells can induce complete HF structures in intact mouse skin in the absence of follicle-derived seen to directly innervate blood vessels by PECAM and PGP 9.5/Gap 43 double stainings. At influences. The disorientation of second-wave HFs in hr/hr skin suggests that in normal skin, the E18, some NFs had penetrated into the epidermis and started to innervate the region between direction of hair growth may be determined by the axis of DP–bulge interactions during early the developing isthmus and bulge of stage 5 tylotrich HFs. These later form the follicular (neural) anagen. Some second-wave HFs in hr/hr skin occasionally grow in the proper direction, thus network B (FNB). Stage 1/2 vellus HFs showed similar innervation and vasculature patterns as producing a second pelage of sparse and faint hairs, which is unable to reverse the overall naked seen in tylotrich HFs at E16. Interestingly NCAM-IR was seen in all stage 1/2 HF keratinocytes appearance of hr/hr skin. By weeks 7–8 pp, the disoriented second-wave HFs disintegrate, leaving but concentrated in keratinocytes of the HF FNB region of stage 5 tylotrich HFs. At P1, HFs behind DP structures which apparently lose their ability to induce another generation of HFs. In from stage 3 possessed singular PGP 9.5-, GAP 43-, and ChAT-IR NFs which encircled the distal contrast, the few secondary HFs that do reach the skin surface and thus re-establish a functional outer root sheath to form the FNA and additional NFs extended towards the FNB region, epithelial-mesenchymal contact leave behind a DP that later on (weeks 9–10) can induce a third, accompanied by S100-IR Schwann cells. The first detectable neuropeptides were substance P (SP) very weak wave of hair growth in hr/hr skin. This suggests that anagen initiation requires not and calcitonin gene related peptide (CGRP), which were located to NFs in the dermis and blood only the DP-derived induction signal, but also a responsive epithelial, perhaps bulge-derived, cell vessels. Aditionally, CGRP was found in epidermal NFs. This study shows, that morphogenesis population. Collectively, our data underscore that hr/hr mice may offer a unique model for of murine skin innervation occurs in successive waves and is strongly correlated to HF and blood dissecting the elusive factors governing the controls of HF cycling. vessel development, further emphasizing a possible interdependence of neuronal and epithelial morphogenesis.

653 654 Exfoliative Cytology as an Innovative Means to Evaluate Epidermal Response to Both Ultraviolet Quantification of Cutaneous Nerves in a Diabetic Mouse Model Using Color Subtractive- Irradiation and Surfactant Exposure Computer Assisted Image Analysis J. Wallner and A. Kligman R. Underwood, N. Gibran,* L. Muffley, M. Usui and J. Olerud SKIN Inc., Conshohocken, Pennsylvania Departments of Medicine (Dermatology) and *Surgery, University of Washington, Seattle, Wash- The purpose of this study was to demonstrate the changes in size, shape and staining properties ington in response to ultraviolet irradiation and injury induced by Sodium Lauryl Sulphate (SLS). Three Immunohistochemistry is a valuable tool for labeling structures of interest in tissue samples, but MEDs of solar simulating radiation was given to the volar forearm. SLS 1% was applied to the the quantification of stained structures has proven difficult. The traditional method of visually other forearm in a chamber for 24 h. Evaluations were made on days 5, 8, 12 and 16. D-squame counting immunohistochemically stained structures is inherently biased, requires multiple observers adhesive discs were used to sample the stratum corneum as a monolayer. These were stained and and generates qualitative data. Stereological methods are complex and labor intensive when evaluated microscopically for corneocyte area, circularity and nuclear presence using an image comparing levels of stain in large numbers of samples. In an effort to quickly, objectively, and analysis program. Other noninvasive techniques include transepidermal water loss (TEWL), reproducibly analyze cutaneous innervation in a diabetic mouse model a Color Subtractive- colorimetry (Minolta), laser Doppler imaging for blood flow. The mean corneocyte area increased, Computer Assisted Image Analysis (CS-CAIA) system was developed. Tissue sections for diabetic peaking between days 12 and 16, for both SLS and UV exposures. Circularity did not change (db/db) mice and their wild type (db/–) litter mates were immunohistochemically stained for the under either condition. With 1% SLS, numerous nuclei were detected at day 5, peaking at day neural marker PGP 9.5 using 3’5’diaminobenzidine as the chromogen. Images were digitally 8, decreasing to near none by day 15. Abundant nuclei were visible by day 8 following UV captured and stored as RGB TIFF files. The brown-red PGP 9.5 staining was colorimetrically exposure but were not quantified due to ill-defined nuclear margins and the appearance of isolated through a scripted process of color background removal using Adobe Photoshop 5.0 and karyorrhexis. TEWL was increased with SLS but not with UV exposure. As expected, the a* IP Tools Plug-in software. The nerve staining was then thresholded and binarized to allow scale of the C.I.E. colorimeter index (Minolta) detected erythema which paralleled visual changes. computer determination of nerve profile counts, area fraction (total area of nerve profiles per unit Tissue perfusion, measured by the laser Doppler scanning, increased shortly after exposure then area of tissue) and areal density (total number of nerve profiles per unit area of tissue). Epidermal decreased to near baseline values by day 5 for both conditions. Exfoliative cytology represents a nerve profile counts, area fraction and areal density were significantly lower in db/db compared noninvasive technique for evaluating injury and repair in acute UV and surfactant exposure. to db/– mice. Although dermal nerve profile counts were reduced in db/db mice compared to Bioengineering techniques produce reliable objective method for assessing the evolution and db/– mice, this reflects the reduction in the dermal area of the db/db mice as there is no significant regression of experimentally induced lesions. difference in the areal density between the two groups. Interobserver reliability of the method was documented by showing agreement between three independent investigators. CS-CAIA not only reduces human labor and bias but also allows stored data to be available for future expanded analysis. 632 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

655 656 Delivery of Oxygen to Cutaneous Tissue Via a Super Saturated Oxygen (SOS) Emulsion Sphingosylphosphorylcholine on Wound Healing: Stimulation of Fibroblast-Embedded Collagen S. C. Davis, T. P. Sullivan, A. L. Cazzangia, E. I. Sanders, W. H. Eaglstein and P. M. Mertz Gel Contraction and Upregulation of Extracellular Matrix Protein Synthesis in Human Dermal Department of Dermatology and Cutaneous Surgery, University of Miami School of Medicine, Fibroblasts Miami, Florida K. Suhr, J. Park, R. Tsuboi and H. Ogawa Oxygen is essential for epithelialization, collagen production and efficient killing of microbes by Department of Dermatology, Chungnam National University, Taejon, Korea; and Department of neutrophils. The purpose of this study is to evaluate the capacity of a super saturated oxygen Dermatology, Juntendo University, Tokyo, Japan (SOS) emulsion containing a perflurocarbon to deliver oxygen to cutaneous tissue. SOS emulsion, Sphingosylphosphorylcholine (SPC) has been known as a wound healing agent due to its potent vehicle and saline were each placed on glass microscope slides. Intact porcine skin with an average mitogenic effect on various cells. The aim of this study is to investigate the effect of SPC on thickness of 0.3 mm was then placed over the glass slides. Viable skin (harvested Ͻ6 h prior to wound contraction and on the synthesis of extracellular matrix (ECM) protein in human dermal the study) and nonviable skin (harvested Ͼ72 h prior to the study) was examined. The epidermal fibroblasts. Using a fibroblast-embedded collagen gel model, we investigated the effect of SPC on surface of the skin was placed in a downward direction and was in direct contact with the collagen gel contraction. SPC stimulated collagen gel contraction in a dose dependent manner, treatment. Radiometer TCM 2 transcutaneous oxygen probes were then secured to the dermal with maximal contraction at 50 mM. Pretreatment with pertussis toxin, suramin, cytochalasin D, side of the pig skin. Initial oxygen readings were taken at 15 min and then every 5 min for 20 staurosporine, or H7 inhibited SPC-induced collagen gel contraction. However, calphostin C, min. There were significant differences in oxygen released from the nonviable pig skin placed genistein or tyrphostin A47 had no effect. Neutralizing antibody against integrin βg1 suppressed over the SOS when compared to nonviable pig skin placed over the other treatment groups: 252 the SPC-stimulated collagen gel contraction, while neutralizing antibodies against TGF-01β1or mmHG O2 (SOS) vs 79 mmHG O2 (saline) vs 33 mmHG O2 (vehicle). Conversely, viable pig PDGF-BB did not abolish the stimulatory effect of SPC on collagen gel contraction. These data skin placed over the SOS did not show statistically significant differences from either vehicle or indicate that integrin 01β1, pertussis toxin-sensitive receptor-and protein kinase-mediated signal saline controls: 57 mmHG O2 (SOS) vs 22 mmHG O2 (saline) vs 26 mmHG O2 (vehicle). transduction are important in SPC-induced fibroblast contraction. The effect of SPC on the Immediately after placement of the viable pig skin over the SOS emulsion the transcutaneous synthesis of ECM protein was also investigated, using immunoblotting and northern blot oxygen levels climbed precipitously but then quickly dropped and leveled off. Whereas, in the hybridization. SPC dose-dependently upregulated the expression of FN protein and mRNA. The nonviable pig skin the oxygen levels remained elevated. The differences in pO2 levels seen in the levels of FN protein and mRNA in SPC-treated fibroblasts were increased 1.8–2.3 times and 1.5– nonviable skin demonstrate that oxygen from the SOS emulsion is able to diffuse through the 2 times, respectively, relative to those in nontreated cells. SPC also upregulated the level of cutaneous tissue. We hypothesize that the lack of a sustained different pO2 level in the viable skin 01α1(1) procollagen mRNA in SPC-treated fibroblasts. Our data indicate that SPC has various reflects utilization of the oxygen provided by the SOS emulsion prior to its reaching the oxygen effect on human dermal fibroblasts, including fibroblast contraction and upregulation of ECM probe. The initial peak in pO2 levels in viable skin may reflect the time needed for the cells protein synthesis. The results of this study further support that SPC has a potential in clinical metabolic pathways to begin utilizing oxygen. The idea of a topical oxygen emulsion to augment application as a wound healing agent. ischemic wound healing is appealing. Studies are being performed to evaluate the effect of the SOS emulsion in healing of ischemic wounds.

657 658 Macrophage-Generated Angiostatin (AS) Requires Macrophase Metalloelastase (MME), but is The Immunohistology of a Human Wrinkle Dependant upon Serine Protease Activity M. Green, W. Parish, A. Kelland, J. Wares, M. Girard, M. Simon* and D. Siegel* S. Raza, L. Nehring, J. Grisolona, D. Kobayashi, S. Shapiro and L. Cornelius Unilever Research, Bedford, UK & Edgewater, New Jersey; and *Departments of Oral Biology Barnes/Jewish Hospital North at Washingtton University School of Medicine, St. Louis, Missouri and Dermatology, SUNY at Stonybrook, New York Matrix metalloproteinases (MMPs) are a family of related Zn-dependent proteases with matrix Wrinkle histology has previously only been examined using chemical stains and without attempt degrading and processing functions. MMPs are released as proenzymes that require activation. to retain the exact details of wrinkle morphology found in vivo. Well defined periorbital ‘‘crows The MMP MME (MMP-12) provides the main MMP protease activity in murine macrophages. feet’’ wrinkles (n ϭ 12) were filled with superglue in situ and, after the glue had set, the wrinkles We have recently shown that activated MME is required for the generation of the angiogenesis were removed and processed for wax histology and confocal microscopy. Sections were examined inhibitor angiostatin (AS) from plasminogen (Plg), and that Plg and plasmin (Pls) stimulate after staining with H&E, orcein or immunostaining for elastin, fibrillin, procollagen types I (PCI) induction and activation of MME from murine macrophages. The serine protease plasmin has & III (PCIII), collagen type I and Factor-VIII. Conventional histology confirmed many previously previously been shown to be an activator, but not an inducer, of MME. To determine the reported observations on these types of wrinkles including collagen alignment across the wrinkle mechanism., we found that the induction of MME was not accompanied by an increase in MME base and an area of relatively photoprotected elastic network at the base of the wrinkle. The mRNA by Northern analysis. Next, an MMP hydroxymate inhibitor was used and effectively degree of photodamage was higher on the lower side of the wrinkle, which would be expected blocked the generation of AS from Plg in MME-competant (MMEϩ/ϩ) macrophages, by western to receive more sun exposure in vivo. Immunohistology supported these observations and in analysis. Interestingly, the serine protease inhibitor aprotinin also blocked AS production. We then addition PCI and PCIII staining was seen to be markedly reduced towards the wrinkle base. used western blot analysis and casein zymography in Plg- and Pls-stimulated macrophages in the Confocal microscopy and triple staining for elastin, fibrillin and factor VIII revealed a complex presence and absence of both inhibitors. Plg and Pls induction and activation of 54 kDa pro- vascular and elastic network. Patterns were consistent with an elastic network joining the base of MME to an active 28 kDa form was inhibited by aprotinin, consequently inhibiting the the wrinkle to the deeper elastic plexus. These data show characteristic marked alterations in the macrophage-mediated generation of AS. Finally, we examined cell-surface expression of MME matrix orientation and composition at the sites of periorbital wrinkles. protein by immunohistochemistry and found that MME-specific antibody positively stained MMEϩ/ϩ, but not MME-deficient (MME–/–) macrophages. Taken together, we demonstrate the novel induction of MME by a serine protease, the dependence of macrophage-generation of AS on plasmin, and possibly support the presence of MME protein on the cell-surface of macrophages

659 660 Nitric Oxide Upregulates both Collagen and Collagenase Synthesis in Cultured Human Dermal Ets Transcriptional Factors Cooperate with Sp1 to Activate the Human Tenascin-C Promoter in Fibroblasts Dermal Fibroblasts M Yamamoto, M Ono,* M Ohtuki, T Moriguchi F. Shirasaki, H. A. Makhluf, D. K. Watson and M. Trojanowska Departments of Plastic Surgery and *Dermatology, Kawasaki Medical School, Okayama, Japan Department of Medicine, Division of Rheumatology and Immunology, and Center for Molecular Nitric Oxide (NO) is a gaseous mediator which is well known as asmooth muscle relaxant and a and Structural Biology, Hollings Cancer Center, Medical University of South Carolina, Charleston, neurotransmitter. It has been shown that NO scavenger impairs the wound healing in animal South Carolina model. Based on these evidences, we hypothesized that NO has a important role as a signaling Tenascin-C (TN-C), an extracellular matrix glycoprotein, is present at low levels in normal adult molecule in the process of wound healing. In this study, we investigated the effect of NO in the tissues, however, elevated expression occurs in wound healing, tumors and fibrotic diseases. To collagen metabolism. Normal dermal fibroblasts were cultured and incubated with orwithout NO better understand the mechanisms of TN-C gene expression, we examined the regulation of the generaters. Then collagen and collagenase expression were analyzed. The result showed that both TN-C gene in human dermal fibroblasts. The TN-C promoter contains multiple consensus sites collagen and collagenase synthesis in human dermal fibroblasts increased by NO at the protein for Sp1 and Ets family transcription factors. The TN-C promoter construct was modestly and mRNA levels. Interestingly, enhancement of collagenase expression was observed at higher transactivated by coexpression with Etsl or Ets2 and strongly transactivated by Fli-1. The Fli-1 NO concentration than that of collagen. These results indicate that NO may act as aregulater of response elements were mapped –133/ϩ75 in the TN-C promoter by deletion analysis. Mutations the collagen metabolism in wound healing. of the four putative Ets binding sites (EBS) located within this region revealed that the site at – 38 mediated Fli-1 induction. In electrophoretic mobility shift assays in vitro translated Fli-1 protein bound to these response elements. Supershift analysis performed with nuclear proteins from human fibroblasts demonstrated that Spl, Sp3 and another member of the Ets family, GABPa, interacted with –38 EBS. To further investigate the relation between Ets and Sp1 in the TN-C promoter, we used Drosophila Schneider cells. Either Ets (Fli-1 or GABPO01αϩ01β) or Sp1 activated the TN-C promoter when added separately and in combination exhibited a synergistic effect. Our data suggest that Fli-1 and Sp1 cooperate to yield higher expression level of the TN-C promoter. Consistent with this hypothesis, recent studies have suggested that during embryogenesis, Fli-1 may be involved in TN-C gene regulation (Int J Dev Biol 39:909–919, 1995). Furthermore, Fli- 1 expression levels in dermal fibroblasts can be induced by several cytokines. From these data, we conclude that Fli-1 might play an important role in regulation of TN-C expression in certain physiologic and pathogenic conditions. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 633

661 662 Co-Localization of EDA-Positive Fibronectin with CD11b (MAC-1)-Positive and CD45 (Leuko- Dermal Defects in Parathyroid Hormone Related-Protein Transgenic Mice cyte)-Positive Cells at the Dermal–Epidermal Junction in Psoriasis Patients by Double-Label J. Gent, C. King, G. Woodard, S. Richardson, E. Farmer and J. Foley Immunohistochemistry Medical Sciences, Indiana University, Bloomington, Indiana; and Department of Dermatology, D. Rothaupt, T. McCormick and K. Cooper Indiana University School of Medicine, Indianapolis, Indiana Department of Dermatology, Case Western Reserve University, University Hospitals of Cleveland, Dermal fibroblasts express the parathyroid hormone/parathyroid hormone related-protein (PTH/ and VA Medical Center, Cleveland, Ohio PTHrP) receptor suggesting that keratinocyte-derived PTHrP may modulate dermal function. Fibronectin (FN) is an extracellular matrix protein which interacts with surface receptors of the Immunohistochemistry, electron microscopy and Western blotting were used to characterize integrin family. It is encoded by a single gene whose premRNA can be spliced in three regions dermal defects in skin samples from newborn, 3-wk and 8-wk-old K14-PTHrP overexpression (EDA, EDB and IIICS). Splicing generates functional differences in fibronectins: growth is mice, rescued PTHrP-knockout mice and their littermates. The dermal alterations of the K14- associated with embryonic FN, stability of the extra cellular matrix is associated with adult FN. PTHrP mice were present at birth and became progressively more apparent in adult animals. An The EDA-positive splice variant of FN is one type of cellular FN which is found during ~3-fold increase in the number of cells was observed in the dermis and this increase was due in embryogenesis. In adults, EDA-FN is normally found in wound repair and angiogenesis of tumor part to increased numbers of nonfibroblast cell types. Large numbers of differentiated melanocytes tissue. However, in uninvolved psoriatic skin, we have previously shown that fibronectin is persisted in the dermis of the adult K14-PTHrP mice. Also, increased numbers of mast cells and involved in induction of proliferation in cooperation with T cell lymphokines, as well as increased capillaries were observed in the upper dermis. Collagen bundles appeared to be less densely packed focal adhesion kinase (FAK) activation and increased cell spreading. The EDA-positive splice and orderly than controls and there were reduced levels of fibronectin protein in the dermis. In variant of FN has been recently identified by our group to be localized at the dermal–epidermal the newborn PTHrP-knockout skin, there was an ~2-fold increase in the number of cells in the junction in psoriasis patients. We now demonstrate by double immunofluorescence staining that dermis, but this increased cellularity dissipated in the 3- and 8-wk-old animals. The dermis had a EDA-FN is colocalized with CD11b and CD45RO-positive cells. Four mm punch biopsies of marked decrease in capillaries as compared to control littermates. The PTHrP-null dermis was involved psoriatic skin, uninvolved psoriatic skin and skin of normal donors were snap-frozen, comprised of tightly packed collagen bundles and the fibroblasts lacked prominent ER and often- cut in 6 µm sections and fixed in acetone. These sections were double-stained for EDA-FN and contained lipid droplets. On the basis of these results, it appears that PTHrP production by the CD45RO or EDA-FN and CD11b. Involved psoriatic skin showed clusters of double-stained epidermis may effect dermal function by altering the cellular composition of the dermis as well as DEJ-lining cells in higher amounts than uninvolved psoriatic skin. Sections from normal donors modulating dermal matrix production. showed only occasional double-stained cells on dermal vessels. CD45RO is expressed on bone marrow derived cells (including monocytes and memory T-cells), whereas CD11b is found mainly on macrophages and granulocytes. Immunocytes are known to play a crucial role in psoriasis. This data demonstrates the close microanatomic localization of EDA-FN and cells of immunologic lineage. Thus, bone marrow derived cells may be involved in the production or assembly of psoriatic EDA-FN at the DEJ-junction, leading to a vicious cycle of extracellular matrix/leukocyte induction of keratinocyte proliferation.

663 664 Colchicine Down-Regulates the Type I Collagen mRNA Expression in Human Dermal Fibroblasts Expression of Antisense Transglutaminase 1 in Fibroblasts Affects Cytoskeletal Organization and K. Chung and D. Kang Cellular Adhesion Department of Dermatology, Yonsei University, Seoul, Korea G. Park, S. Kee, S. Kim and P. Steinert Colchicine is primarily an antigout drug obtained from species of Colchicum. In addition to the Laboratory of Skin Biology, NIAMS, NIH, Bethesda, Maryland antigout effect, it has been found to have weak anti-inflammatory activity and also to decrease The function of the transglutaminase 1 (TGase 1) enzyme is well understood in epithelial systems: the synthesis of collagen in fibroblasts. Due to the latter effect, it is used as an antifibrotic agent at least one major role is crosslinking of substrates to make the cornified cell envelope. In addition, in the treatment of sclerotic diseases such as scleroderma, liver cirrhosis and primary biliary fibrosis. we have discovered that TGase 1 is expressed in normal human dermal fibroblasts (NHDF) at Previous studies have shown that the effect of colchicine on collagen is post-translational by which both the mRNA and protein levels in about 1%–10% of the amount in the epidermis. However, the excretion of collagen from fibroblasts is inhibited by down-regulating the formation of the function of TGase 1 in NHDF is unknown. In this study, we constructed antisense probes in intracellular microtubules. Therefore, the mechanism of collagen inhibition has been known to the pCI-neo vector of TGases 1 and 2 using reverse sequences encoding their amino-termini (1– be due more likely to an inhibition of cellular collagen secretion than to a switch off of collagen 347 bp and 1–397 bp, respectively). The constructed plasmids were then transfected into NHDF. mRNA transcription. We have added colchicine to in vitro culture of human neonatal foreskin After 48 h, we confirmed the expression of the antisense TGase 1 and 2 vectors using RT-PCR, dermal fibroblasts and checked the expression of type I collagen mRNA by Northern blotting to and also we observed reduced total TGase activity. NHDF transfected with antisense TGase 1 verify if the drug has any effect on the transcriptional stage of type I collagen synthesis. Interestingly, were greatly reduced in size and many cells had detached from the surface of the plastic culture in contrast to previous studies, colchicine clearly down-regulated the basal level of type I collagen flask. NHDF transfected with antisense TGase 2 did not significantly affect cell shape or adhesion. mRNA expression and also counteracted the stimulatory effect of TGF-01β. It can be concluded We also analyzed the organization of several cytoskeletal proteins using immunofluorescence from this study that colchicine, in addition to its already known role as a post-translational staining. NHDF transfected with antisense TGase 1 showed translocalization of 01β-tubulin, regulator of type I collagen synthesis, can also influence the mRNA expression, thereby down- vimentin and TGase 2 to the perinuclear region, but actin organiztion was not changed. In NHDF regulating the synthesis of type I collagen presumably at the transcriptional level. transfected with antisense TGase 2, there were no noticeable changes in cytoskeletal protein organization in 72 h after transfection. Together, these results suggest that TGase 1 may play key roles in the organization of the cytoskeleton and cellular attachment in fibroblasts.

665 666 Increased Expression of Transglutaminases 1 and 2 Colocalizes With Amyloid Deposition in Short Wavelength UVB (290–300 nm) and Long Wavelength UVA (360–400 nm) Most Efficiently Muscle of Inclusion Body Myositis Induce Collagenase in Human Skin In Vivo S. Kim and P. Steinert and Y. C. Choi* G. Fisher, Z. Wang, H. Choi, S. Kang and J. Voorhees Laboratory of Skin Biology, NIAMS, NIH, Bethesda, Maryland; and *Department of Neurology, Department of Dermatology, University of Michigan, Ann Arbor, Michigan College of Medicine, Yonsei University, Seoul, Korea Ultraviolet (UV) irradiation induces collagenase and other matrix-degrading metalloproteinases Inclusion body myositis (IBM) is the most common muscle disease in persons Ͼ50-y-old. It is that damage dermal collagen and elastin, leading to sun-induced premature skin aging. We have characterized by abnormally accumulated amyloid protein within the vacuolated muscle fibers, investigated the UV wavelength dependence for induction of collagenase in human skin in vivo. which have been implicated in the cause of IBM. Recent studies have documented increased We utilized three UV sources: (i) UVB fluorescent tubes, (ii) UVB ϩ UVA fluorescent tubes in expression of transglutaminases (TGases) 1 and 2 in Alzheimer’s disease, and thus TGases might combination, and (iii) Xenon arc solar simulator. Each of these UV sources was filtered to block be involved in the pathogenesis of 01β-amyloid deposits and increases in neurotoxicity. By analogy, wavelengths below 290 nm (UVC). The UV spectral output of each source was; UVB tubes, we have wondered whether IBM disease might also be correlated with increased TGase expression, 63% UVB, 25% UVA2, and 12% UVA1; UVB ϩ UVA tubes, 11% UVB, 25% UVA2, and 64% but the expression of TGases in muscle and their possible participation in the pathogenesis of UVA1; solar simulator, 7% UVB, 16% UVA2, and 77% UVA1. Exposure of human skin to 2MED muscle diseases has not been explored. In this study, we have investigated the levels of expression from each of these three sources yielded similar induction of collagenase mRNA (8–10-fold). of TGases using RT-PCR, enzyme activity assays, and immunofluoresence staining of normal and Reducing by 20-fold short wavelength (290–300 nm) output from the UVB tubes (by cutoff IBM tissues. In normal muscle tissues, TGase 1 is undetectable, and TGase 2 decorates the filter) reduced collagenase mRNA induction by 80%, in response to a 2MED exposure (80 mJ endomysium of muscle fibers. Interestingly, we found that total TGase activity in IBM tissue was per cm2). To determine the relative capacity of UVA wavelengths (320–400 nm) to induce increased up to 20-fold in comparison to normal human tissues, and mRNA levels of TGases 1 collagenase, we utilized a series of five cutoff filters that successively blocked shorter UVA and 2 were increased about 30% and about 2-fold, respectively. Furthermore, we found bright wavelengths emitted from the UVB ϩ UVA source. UV exposures through each filter were colocalization of TGases 1 and 2 with the 01β-amyloid precursor and 01β-amyloid protein in the constant at 3.5 J per cm2. At this UV dose, pure UVA1 (360–400 nm) most efficiently induced vacuoles of IBM muscle fibers. It is known that metalloprotease expression is elevated in the collagenase (4.5 Ϯ 1.3-fold, N ϭ 7). Our data indicate that UVB is approx. Fifty times more vacuoles of IBM muscle. Thus it is possible these proteases may also activate the abnormally potent than UVA1 in inducing collagenase in human skin in vivo. We conclude that both short expressed TGase 1. Accordingly, we conclude that TGases 1 and 2 are abnormally accumulated wavelength UVB and UVA1 can induce MMPs and cause photoaging. Therefore, sunscreens that in vacuolated muscle fibers of IBM and might have important roles of amyloid deposition. block only UVB may promote photoaging from UVA1 by allowing longer sun exposures without sunburn. 634 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

667 668 Ultraviolet Irradiation Induces Metalloelastase in Human Skin In Vivo Reduced Collagen Production by Fibroblasts in Photoaged Skin In Vivo is Normalized in Cultured J. Chung, J. Lee, S. Kang, Z. Wang, G. Fisher and J. Voorhees Fibroblasts From Photoaged Skin Department of Dermatology, University of Michigan, Ann Arbor, Michigan J. Varani, Z. Wang, P. Perone, G. Fisher and J. Voorhees Chronic exposure to ultraviolet (UV) irradiation causes degenerative alterations in collagen and Departments of Pathology and Dermatology, University of Michigan, Ann Arbor, Michigan elastin in the dermal extracellular matrix. This damage, which is the hallmark of photoaged skin, Reduced collagen content is a characteristic feature of photoaged skin. Increased levels of collagen- is mediated in large part by UV-induced matrix metalloproteinases (MMP). Metalloelastase (MMP- degrading matrix metalloproteinases in response to solar UV irradiation are, in part, responsible 12) is a member of the matrix metalloproteinase family that specifically degrades elastin. We have for this, but decreased collagen production may also contribute. The question addressed in the investigated expression of metalloelastase in UV-irradiated human skin in vivo. UV (UVB source present study is whether collagen production is decreased in photoaged skin and if it is, does the filtered to remove UVC) irradiation induced expression of metalloelastase mRNA in a dose- decrease reflect irreversible injury to the dermal fibroblasts. To address this question, replicate 4- dependent manner. Metalloelastase mRNA was induced 7.7-fold (N ϭ 3) by 2MED. Maximal mm punch biopsies were obtained from forearm skin of individuals with extensive sun-damage. induction of metalloelastase mRNA occurred 16 h post UV. Pretreatment of skin with the Biopsies of underarm and hip skin were obtained from the same individuals for comparison. Based antioxidants N-acetyl cysteine (NAC, 20%) or Vitamin E (5%) inhibited metalloelastase expression on 14C-proline incorporation into pepsin-resistant material and in situ hybridization for 01α1(I) 54% and 47% (N ϭ 4), respectively, in human skin in vivo. A major source of metalloelastase is procollagen mRNA as endpoints, collagen synthesis was reduced in forearm skin as compared to macrophages. Whether keratinocytes or fibroblasts can also synthesize metalloelastase has not been skin from the other two sites by 36% (n ϭ 9; P Ͻ 0.05) and by 51% (n ϭ 7; P Ͻ 0.05), reported. Therefore, we exposed cultured human keratinocytes and dermal fibroblasts to UV (30 respectively. In parallel, biopsies from each site were cut into fragments (15–20 per biopsy) and mJ per cm2) and determined metalloelastase mRNA levels by northern blot. UV induced used for isolation of fibroblasts. Fibroblasts were isolated from a comparable percentage of fragments metalloelastase mRNA in human dermal fibroblasts. No metalloelastase mRNA was detected in from all three sites (26, 21 and 32%, for forearm, underarm and hip, respectively) (n ϭ 6). After nonirradiated or UV-irradiated human keratinocytes. These data demonstrate that UV induces expansion, fibroblasts from the three sites produced virtually identical amounts of collagen (ratio metalloelastase in human skin in vivo, and cultured dermal fibroblasts. Metalloelastase likely of 1.0:1.3:0.8 for forearm, underarm and hip; n ϭ 12, 13 and 20, respectively). These studies contributes, along with other UV-induced MMPs such as gelatinase B and stromelysin, to elastin indicate that collagen synthesis is decreased in badly photoaged skin as compared to sun-protected degradation in human skin. Dermal fibroblasts, in addition to infiltrating macrophages, may be a skin. The reduced collagen production in photoaged skin is evidently related to the in vivo context source of increased metalloelastase in UV-irradiated human skin. Antioxidants would be expected because fibroblasts from photoaged skin produced normal amounts of collagen in vitro. to lessen UV-induced elastin damage, by inhibiting metalloelastase induction, and thereby protect against photoaging.

669 670 Age-Related Changes in Skin Proteoglycans Antisense MT1-MMP cDNA Expression in Human Keratinocytes and Dermal Microvascular D. A. Carrino, J. Sorrell, M. Seavolt, M. Baber and A. Caplan Endothelial Cells Department of Biology, Case Western Reserve University, Cleveland, Ohio U. Nagavarapu, S. Wasan, B. Gardner, J. Yang, K. Relloma and G. Herron Besides collagen, another major class of extracellular matrix molecules is proteoglycans (PGs), Department of Dermatology, Stanford University, Stanford, California which are glycoconjugates consisting of a core protein and covalently attached carbohydrate in Matrix metalloproteinases (MMPs) are a family of at least 15 secreted and membrane-bound zinc the form of glycosaminoglycans (GAGs). Because PGs have been shown to affect tissue hydration, endopeptidases involved in multiple proteinase activation cascade systems in a variety of different collagen fibrillogenesis, and cell migration, PGs from full-thickness human skin of different ages tissues and cell types. Membrane type MMP-1 (MT1-MMP or MMP14) expression by fibroblasts, were isolated and compared in order to assess whether age-related changes occur in the production endothelial cells and tumors is believed to play a crucial role in matrix remodeling during cellular of these molecules. Three types of PGs are found in abundance in these extracts: versican, a large migration and invasion. Overexpression of MT1-MMP mRNA has been reported in many tumors chondroitin sulfate PG, decorin, a small, collagen-binding dermatan sulfate PG, and a truncated and is correlated with activation of MMP-2. In this study, we characterize the effects of antisense form of decorin. Versican is most abundant in fetal skin and represents only a small proportion of MT1-MMP gene expression in normal human keratinocyte and endothelial cells. Retroviral the PGs in postnatal skin for the ages examined (26–82 y). In contrast, the relative amount of constructs bearing a 5Ј 1.6 kb antisense construct decreased both MT1-MMP protein and mRNA decorin increases as a function of age, and there are age-related changes in the size of decorin. expression in primary keratinocytes and endothelial cells. HPVE6/E7-immortalized keratinocytes The core protein of decorin shows age-related differences in polydispersity, which appear to be and telomerase-immortalized endothelial cells showed a similar effect. The reduction in protein due in part to differences in asparagine-linked oligosaccharides. Postnatal skin also contains a and mRNA expression in all cell types was optimal 48–72 h after antisense transduction. truncated decorin, which is identified as such based on amino-terminal amino acid sequence Zymographic analysis of media conditioned by MT1-MMP antisense-transduced cells treated with analysis and immunoreactivity with antibodies to decorin core protein. This molecule has a core conconavalin A revealed inhibition of proMMP2 activation. MT1-MMP antisense expression did protein approximately half the size of that of decorin (20 vs 45 kilodaltons). The GAGs of the not correlate with major changes in cell morphology or growth in vitro. In summary, antisense truncated decorin appear to differ from those of decorin, based on reactivity with a monoclonal MT1-MMP expression provides a rapid and efficient method for modulating MMP activities antibody which recognizes the GAGs of the latter but not of the former. This makes less likely in vitro and may help to clarify the functional role of MT1-MMP activity in cutaneous extracellular the possibility that the truncated decorin arises by catabolism of decorin and suggests the possibility matrix metabolism. that it is a splice variant. The age-related changes observed in human skin PGs are not related to anatomic site, since skin from several sites shows similar results. These data indicate that human skin shows age-related changes in PGs.

671 672 Quantitative Measurement of Matrix Metalloproteinase Activities Using Fluorogenic Substrates: Cloning of Human Laminin 01α5 Chain: Expression in Normal Epidermis, Melanoma, Basal Cell Keratinocyte Proteinase Expression Carcinoma and Squamous Cell Carcinoma Y. Ao, S. Hwang, K. Relloma and G. Herron J. Li, H. Tran, C. Chui, M. Koch and P. Marinkovich Department of Dermatology, Stanford University, Stanford, California Harvard University of Boston, Massachusetts; Stanford University, Palo Alto, California Few rapid and reliable methods for quantitating matrix metalloproteinase (MMP) activities in Laminins are important components of all basement membranes and influence cell attachment and biological samples are currently available. In this study, we characterize an assay system for MMP migration. The goal of these studies was to identify a poorly characterized laminin present in skin. activities based on recently developed fluorogenic substrates which utilize 7-methoxycoumarin In addition, to laminins 5 and 6, previous studies show that keratinocytes produce a laminin (MOC)-labelled MMP-specific small peptides (Knight, FEBS Lett 296:263). Release of MOC containing a 01β1 chain, a 01γ1 chain, and an approximately 400 kDa a chain. We identified this from the 2,4-dinitrophenyl-quenched peptide by active MMPs results in proportional increases in 01α chain to be laminin 01α5 chain by immunoblot of keratinocyte and squamous carcinoma fluorescence with time. Activities of recombinant MMP-2 and MMP-9 using the ‘‘Knight’’ cell (SCC) extracts using a specific a 5 chain polyclonal antibody (Ab). In addition, we show, substrate were readily detectable and yielded linear initial reaction rates over 10 min. Calculated using polyclonal and monoclonal Abs to human laminin 01α5 chain by IDIF microscopy significant initial rates of hydrolysis of the Knight substrate (4 µM) by MMP-2 (1.2 nM), MMP-9 (1.2 nM), expression of this chain in the basement membrane of frozen sections of normal human skin, and MMP-2 (0.6 µM) plus MMP-9 (0.6 µM) were 571.1 units per min, 208.2 units per min, SCC tumors, basal cell carcinomas and primary/metastatic melanomas. As a first step to determine and 242 units per min, respectively. Activity measurements of conditioned media (CM) from the function of human laminin 01α5 chain in normal and neoplastic tissues, we sought to obtain primary normal human (NHK) and immortalized keratinocyte (NIK) cultures showed low net full-length sequence of the human laminin a 5 cDNA. As the cloning of the C terminus has been MMP activity detectable only in PMA-treated NHK and only after APMA activation (10.1 units accomplished, we sought to isolate N terminal cDNA clones. Two human clones were identified per min per ug total protein). Values for concentrated CM from NIK cultures Ϯ PMA were 0.22 with 80% homology to the N-terminus of mouse laminin 01α5 chain. With addition of these units per min per ug total protein and 0.32 units per min per total protein, respectively. Reverse clones to those which have already been identified, only approximately 2 kB of the 11 kB zymography confirmed that gelatinase activities in CM from NHK were much higher than from predicted human alpha 5 sequence remains to be identified. NIK, that APMA converted proenzymes to active forms and that TIMP-1 was increased in NIK CM. Stromelysin activity was also measured using an MMP3-specific fluorogenic substrate (Nagase, JBC 269:20952). Initial rates for MMP3 were detectable only in concentrated NIK media Ϯ PMA at 0.47 units per min per ug total protein and 1.1 units per min per ug total protein, respectively. We conclude that the fluorogenic substrate assay is a fast and reproducible method for quantifying net MMP activities in CM; however, TIMPs appear to significantly decrease the sensitvity of this assay system and thus it should always be used in combination with zymography and/or after removal of TIMPs. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 635

673 674 Delineation of Genes Expressed in Human Epidermis by Large-Scale Sequencing of a cDNA A Biologically Active DR5 Type of Retinoic Acid Response Element (RARE) Controls Human Library Generated from Laser Capture Microdissected Skin Transglutaminase Type 1 Gene Promoter Activity in Epidermal Keratinocytes T. Darling, B. Koh and K. Yancey P. LaCelle, M. P. Santini, B. Graf and R. Polakowska Dermatology Branch, NCI, National Institutes of Health, Bethesda, Maryland University of Rochester School of Medicine, Rochester, New York, New York Large-scale sequencing of cDNA libraries can profile relative levels of gene expression and identify Expression of the transglutaminase type1 gene (TGM1), which encodes an epithelial cell-specific novel genes. Prior studies have used this method to examine cultured keratinocytes but not protein-crosslinking enzyme, is limited to particular stages of epidermal development and epidermis in its native state. In the current study, recently developed technologies were used to keratinocyte differentiation. As a result, transglutaminase type 1 enzyme (TGase) activity in profile the expression of known and unknown genes in normal human epidermis. Epidermis from epidermal cells increases with the onset of keratinization in vivo and in vitro. Retinoic acid (RA) 20 cryostat skin sections (8 µm thick) was selectively isolated using laser capture microdissection. inhibits terminal differentiation of cultured keratinocytes and downregulates the steady-state levels Total RNA extracted from the microdissected epidermis was reverse transcribed using novel of TGM1 mRNA. We determined, by functional mapping of deletion mutations in the TGM1 primers permitting PCR amplification and ligation-independent directional cloning of cDNAs 5Ј untranslated region, that the 5Ј upstream regulatory region of the proximal promoter contains into the pAMP1 vector with uracil DNA glycosylase. Clones from this library were subjected to 5’Xho1/HindIII elements instrumental in the suppression by RA. Inspection of the 5’X/H high throughput automated sequencing and BLAST sequence similarity searches were performed sequence identified a direct repeat 5 (DR5) retinoic acid response element (RARE) potentially against the Genbank/EMBL/DDBJ databases. Of sequences evaluated to date, ~70% matched mediating the retinoic acid receptor (RAR)-signaling. The functional significance of this RARE known genes, including the differentiation markers loricrin and keratin 1. Sequences corresponding sequence in keratinocytes was established by DNA-protein binding and transactivation assays to ribosomal proteins were frequently represented in epidermis, a finding observed in other tissues demonstrating that the TGM1 gene promoter contains a biologically active RAR/RXR heterod- with a high proliferative rate. Another ~20% of the cDNAs matched expressed sequence tags imer binding site. (ESTs) from other tissues. Approximately 5% did not correspond to any previously known sequence, suggesting that these may represent novel genes. These studies will elucidate the pattern of gene expression in human epidermis in vivo, and provide molecular tools for identifying genes important in epidermal biology and human skin diseases.

675 676 Genetic Regulation of the Fucosyltransferase VII (Fuc TVII) Gene in Leukocytes cGMP Gated Channel and Alternatively Spliced Forms in Keratinocyte Differentiation A. M. Chapas, B. E. Rich and T. S. Kupper Y. Oda, U. Lee, C. Largman and T. Mauro Harvard Skin Disease Research Center, Brigham and Women’s Hospital, Boston, Massachusetts Departments of Dermatology, University of California San Francisco, VA Medical Center, San Human T lymphocytes expressing the surface glycoprotein known as cutaneous lymphocyte Francisco, California antigen (CLA) preferentially home to the skin. CLA is defined by the mAb HECA-452, which The terminal differentiation of keratinocytes is induced by raised extracellular calcium. Previously, recognizes an oligosaccharide epitope. Recent data indicates that the expression of CLA is regulated we have cloned a cGMP gated channel from human keratinocytes (CNG1) which is permeable by the differential glycosylation of an already existing surface protein, P-selectin ligand glycoprotein- to calcium. Both the full length channel and three alternatively spliced forms were detected in 1 (PSGL-1). The enzyme, fucosyltransferase VII (Fuc TVII), performs a key step in CLA formation keratinocytes. Application of cell-permeant cGMP and nitroprusside enhanced keratinocyte by adding fucose to the terminally sialylated lactosamine motifs on PSGL-1. To understand how differentiation, as measured by the expression of involucrin, suggesting a role for these channels Fuc TVII is regulated, 1.1 kilobases of genetic sequence upstream from its transcription start site in keratinocyte differentiation. In early stages of differentiation, only the inactive spliced variant, were cloned and sequenced. This sequence contains a number of candidate transcription factor lacking both the N-terminal intracellular and first transmembrane domain (CNG1–4), was binding sites, including several NF01κB-IL2Ra binding regions, AP-2 binding sites, and a TATA expressed. The functionally active full length channel appeared only after the cells differentiated. box. To directly evaluate whether this upstream sequence could drive Fuc TVII gene expression, The spliced variant (CNG1–4) also coexpressed with the full length channel. All four forms of the sequence was inserted in front of the luciferase gene of a reporter plasmid. Transfection of the channel produced channel proteins when they were in vitro transcribed and translated in this reporter construct into U-937 cells, a histiocytic lymphoma cell line known to constitutively reticulocyte lysates. However, only the full length channel expressed a functional channel protein express Fuc TVII, yielded greater than 6-fold relative luciferase units (RLUs) than the control when cDNAs of these channels were transfected into mammalian cells (HEK293). Cotransfection plasmid. Analysis of a series of 5Ј deletion plasmids will elucidate the minimal region necessary to of the variant (CNG1–4) blocked the normal expression of the full length channel when the ratio drive Fuc TVII transcription. Using RT-PCR, we have characterized the conditions that activate of the variant to the full length channel exceeded 1:10. These results suggest that the cGMP gated Fuc TVII transcription in Jurkat cells. Jurkat cells treated with 5 nM PMA or anti-CD3 mAbs channel might regulate its role in keratinocyte differentiation by manufacturing varying proportions showed increased transcription of the Fuc TVII after 4 h of treatment. Ongoing analysis of Jurkat of the full length and alternatively spliced variants. cells expressing the reporter construct will further define the kinetics of Fuc TVII activation. Thus, we have identified upstream regions sufficient for Fuc TVII gene transcription, and have identified potential transcription factors that interact with this region to regulate this enzyme’s activity. Ideally, this knowledge will help to suggest novel approaches to treating T cell mediated inflammatory skin diseases

677 678 Withdrawn Versican Promoter Transactivation in Dermal Papilla as a Marker for Hair Inductive Activity: Novel Approach to Isolate Fresh Interfollicular Dermal Papilla Cells J. Kishimoto, R. Ehana and R. Burgeson Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard, Boston, Massa- chusetts We have previously reported that the promoter of versican, a large chondroitin sulfate proteoglycan, exhibits hair dermal papilla (DP) specific transcriptional activation of a lacZ reporter gene in transgenic mice (Kishimoto J. et al., J Invest Dermatol 110:497, 1998). To examine whether this gene activation correlates with hair inductive ability by DP cells, versican positive DP cells were isolated for hair grafting assay. To this end, another transgenic line in which green fluorescent protein (GFP) is expressed under versican promoter control was generated so that versican positive cells could be selected based on the GFP fluorescence. Resultant versican-GFP transgenic mouse exhibited GFP fluorescence in DP. No GFP expression was observed either in epidermis or epithelial components of the hair follicle. Endogenous mouse versican mRNA expression in DP was also confirmed by in situ hybridization. Newborn skin from this transgenic mouse was dissected and the dermis was dissociated with collagenase. After removal of undissociated preformed follicle pellets, the dermal cell suspension was separated into GFP positive (ϭ DP cells) and GFP negative (dermal fibroblasts) cell pools using a high-speed cell sorter. Each pool containing 6 01ϫ 106 cells was grafted onto nude mice back skin together with murine keratinocytes, and hair growth was examined 3 weeks after grafting. The results clearly showed that only grafting of GFP positive cells resulted in hair growth (five of five grafts), whereas GFP negative cells did not (none of six grafts). Histological evaluation of the grafting site revealed that DP cells in reconstituted follicles re-exhibited GFP fluorescence. Also, cultured GFP positive cells lost fluorescence following cell division whereas DP cells cultured within preformed follicles showed prolonged GFP fluorescence. These results suggest that epithelial derived factor(s) are required for transcriptional activation of versican gene expression and imply that versican expression may be essential for hair inductive ability of DP cells. This is a first demonstration of the isolation of large numbers of fresh interfollicular DP cells at the time which they have hair inductive potency, and these GFP fluorescent cells should be a useful tool to further characterize DP cells. 636 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

679 680 Cloning and Characterization of Retinol Dehydrogenase Transcripts Expressed in Human Cloning, Sequencing and Overexpression of the Propionibacterium acnes dnaK (Heat Shock Protein Epidermal Keratinocytes 70) Homologue N. G. Markova,* V. Jurukovski,* N. Karaman-Jurukovska,* R. K. Randolph,* J. Su,† J. Napoli† M. D. Farrar, E. Ingham and K. T. Holland and M. Simon*‡ Skin Research Centre, Department of Microbiology, University of Leeds, Leeds, UK *The Living Skin Bank, Department of Oral Biology and Pathology, School of Dental Medicine, Heat shock proteins are dominant antigens of many bacteria and have been implicated in and ‡Department of Dermatology, School of Medicine, SUNY at Stony Brook, Stony Brook, inflammatory processes. To test the hypothesis that these proteins are involved in inflammatory New York, †Department of Biochemistry, School of Medicine and Biomedical Sciences, SUNY acne vulgaris it was essential to confirm the presence of heat shock gene homologues in P. acnes at Buffalo, Buffalo, New York and to then clone and overexpress these genes in order to obtain sufficient protein for immunological The normal growth and differentiation of the epidermis requires an adequate supply of vitamin investigation. A fragment of the P. acnes dnaK homologue was amplified by PCR using degenerate A. The active form of vitamin A for normal epidermal homeostasis is retinoic acid (RA). Retinoic primers coding for highly conserved regions of DnaK proteins of other bacteria. This fragment acid controls the expression of retinoid-responsive genes via interactions of the retinoic acid/ was used as a probe to isolate the whole dnaK gene from a bacteriophage l library of P. acnes nuclear receptor complexes at specific DNA sequences in their control regions. The message genomic DNA. The whole gene was cloned into the expression vector pET-30b under the conveyed by RA is likely modulated by the concentration of the ligand available for binding to control of the bacteriophage T7 promoter and DnaK overexpressed with a C-terminal histidine the receptors. Following the uptake of plasma retinol, epidermal keratinocytes synthesize retinoic tag in Escherichia coli. The dnaK gene of P. acnes encodes a protein of 617 amino acids with a acid via two sequential reactions with retinaldehyde as an intermediate. Several retinol dehydro- predicted molecular weight of 66.4 kilodaltons. The protein shows a high degree of homology genase (RDH) enzymes, members of the short-chain dehydrogenase/reductase (SDR) gene to DnaK proteins of other bacterial species, particularly those of mycobacteria (e.g. 67% identity, superfamily, catalyze the first and rate-limiting step that generates retinaldehyde from retinol bound 79% similarity to Mycobacterium tuberculosis DnaK) which cause chronic inflammation in humans. to cellular retinol binding protein (holo-CRBP). However, little is known about these enzymes P. acnes possesses a DnaK homologue which shows high homology to other bacterial DnaK and their genes in the epidermal cells. Our work describes the first member of the RDH family proteins and will now be used to determine its presence in inflamed follicles. found in epidermis. We show that this gene is expressed predominantly in the differentiating spinous layers and that it is under positive, feed-forward regulation by retinoic acid. It encodes a protein that, using NADϩ as a preferred cofactor, utilizes free-and CRBP-bound all-trans-retinol, and steroids as substrates.

681 682 Glucose Inhibits Induction of Vascular Endothelial Cell Growth Factor by Hypoxia Does a Conformational Change of the Promoter Region Repress Human Loricrin Expression? R. A. Swerlick, K. Casper and S. Naik S. Jang, M. Morasso, A. Rossi* and P. Steinert Department of Dermatology and Emory University School of Medicine, Atlanta, Georgia Laboratory of Skin Biology, NIAMS, NIH, Bethesda, Maryland; *Department of Experimental Vascular endothelial cell growth factor (VEGF) is an important vascular growth and survival factor. Medicine, University Tor Vergata, Rome, Italy VEGF expression is complex and regulated by multiple stimuli, including oxygen tension and In the epidermis loricrin is expressed in the granular layer and is subsequently cross-linked by glucose. Previous studies have demonstrated that either hypoxia or hypoglycemia results in transglutaminases with SPRs, involucrin, and keratins to form the cornified cell envelope. The induction of VEGF mRNA. Furthermore, hyperoxia may inhibit VEGF production and result in expression of loricrin is tightly controlled during the terminal differentiation in keratinocytes. vascular regression. Studies have not examined the effects of mixed signals such as hyperglycemia Previously we identified three positive motifs of Sp1 (–125), CRE-like (–100) and AP1 (–60) and hypoxia, a state relevant to diabetes. We examined whether glucose, in concentrations relevant located in the proximal promoter region in the first 160 bp from the transcription start site. We to tissue concentrations in poorly controlled diabetes, may blunt induction of VEGF gene have shown that the AP1 site is essential for both transcriptional activity and calcium responsiveness. expression in response to hypoxia. Human dermal fibroblasts (HDF) were exposed to hypoxic In this study, we have identified a possible mechanism for the control of human loricrin. We conditions (0.5% O2) in the presence of normoglycemia (100 mg per dl glucose) or hyperglycemia found four negative elements with sequences of (A/G)GGCCA, namely R1 (–150), R2 (–110), (450 mg per dl) for 1–24 h and examined for VEGF mRNA by northern blot. HDF exposed to R3 (–90) and R4 (–70) located in the proximal promoter region interspersed with the positive hypoxia under normoglycemic conditions demonstrated increased VEGF mRNA expression that elements. Notably, each the four negative elements are exactly separated by two or four turns of was detected by 6 h and maximal after 48 h. However, HDF exposed to hypoxia in the presence the DNA helix. Results from competition assays showed that a yet-to-be identified protein caused of hyperglycemia demonstrated a markedly blunted VEGF response which, although detectable gel-mobility shifts when each element was separately incubated with nuclear extracts from normal after 24 h, disappeared after 48 h. The inhibition of VEGF mRNA induction was concentration- human epidermal keratinocytes (NHEK). Mutation of the R2 site led to a 9-fold increase in CAT dependent and evident at glucose concentrations as low as 200 mg per dl, and maximal at 300– activity in NHEK but not in HeLa or primary human fibroblasts. Mutation of the R3 or R4 sites 450 mg per dl. The effect of glucose was not cell-specific, and was also observed in the epithelial resulted in a 6-fold increase in NHEK and 4-fold increase in HeLa and human fibroblasts. Thus cell lines HeLa and A431. In contrast to HDF, VEGF mRNA expression in HeLa and A431 was of these four elements, R2 is critical for keratinocyte-specific expression. Since an activating Sp1 induced after 1 h of hypoxia, but induction of VEGF mRNA was also inhibited by glucose in motif is very close to the R2 site, we wanted to examine whether these two competing elements a concentration-dependent fashion. These data support the hypothesis that elevated glucose interact. The Sp1 and R2 sites were replaced by a Gal4 binding sequences and was cotransfected concentrations in diabetes may blunt physiologic responses to tissue hypoxia and play a role in with Gal4-Sp1 expression plasmids. Results showed that the Sp1 transactivation domains A, B, C diabetes-related end-organ damage but not D caused a 5-fold increase of loricrin promoter activity in NHEK. Together these data suggest that the binding on these four negative (A/G)GGCCA elements may function by changing the DNA conformation to prevent the activating AP1, CREB and Sp1 proteins from binding to their motifs.

683 684 Cloning of a Novel, Ubiquitously Expressed Yemanuclein-Like Nuclear Protein (YEL) with Transcriptional Regulation of Distal-less 3 (Dlx3) Homeobox Gene upon Differentiation of Features of a Transcriptional Regulator Mouse Keratinocytes S. Aho, Y. Ryoo, M. Buisson, J-F. Giot, A. Sergeant, B. Cho, K. Rothenberger and J. Uitto M. Morasso and G. Park Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Philadelphia, Laboratory of Skin Biology, NIAMS, NIH, Bethesda, Maryland Pennsylvania; Laboratoire de Virologie Humaine, U412 INSERM, ENS-Lyon, France The Dlx3 homeodomain gene is expressed in terminally differentiated murine epidermal cells and We have identiﬁed a novel gene, YEL, located in tail-to-tail orientation in a close proximity with is a crucial regulator of late differentiation markers of keratinocytes. In an attempt to determine the periplakin gene in human chr 16p13.3. A total of 18 exons span ~70 kb of genomic sequences the upstream regulators of Dlx3, we have cloned a 1.2-kb proximal region of the murine gene. and give rise to a 7-kb transcript. Analysis of the cDNA sequence revealed an open reading frame The proximal region of Dlx3 gene has a canonical TATA box and CAAT box. Serial deletion of 3402 bp, predicting a 121 529 Da protein, which is basic (pI 9.34) and serine rich (12.79%). analysis showed that the region between –84 and –34 confers the maximal promoter activity in A partial cDNA of YEL (VT4), encoding aa 348–692, has been cloned on the basis of its primary keratinocytes as well as in other epithelial cell types such as Hela cells. Gel retardation interaction with a negative response element (NRE1) of HIV-1 LTR, and another partial cDNA assays and mutational analysis demonstrated that the CAAT box, which is located at position –74 (ZAP5), encoding aa 248–1134, was cloned through an interaction with EBV immediate early and is responsible for the majority of the activity of the promoter. By super-shift analysis with protein EB1/Zta. ZAP5, when overexpressed in HeLa cells, showed nuclear localization, and was speciﬁc antibodies for distinct CCAAT-binding proteins such as C/EBP, NF-1 and NF-Y, we shown in vitro to interact with the basic domain of transcription factor c-Jun and with c/EBP and showed that the NF-Y factor (also referred to as CBF) binds to the CCAAT site in nuclear to regulate the expression of cellular genes, including periplakin. Multiple repeats of basic amino extracts of undifferentiated and differentiated mouse primary keratinocytes cultured in vitro.We acids showed similarity to known nuclear localization signals. The amino-terminal end of YEL also found a canonical Sp1 binding site located immediately upstream of transcription start site at showed considerable homology to yemanuclein-01α, a maternally expressed Drosophila transcript. position –13, that acts as a positive regulatory element of the Dlx3 promoter. Super-shift assays The acidic domain, aa 480–560, has similarity to the acidic domains of yemanuclein-01α and with antibodies against Sp1, Sp2, Sp3 and Sp4, showed that Sp1 and Sp3 participate in the nucleolin. PCR analysis of Multiple Tissue cDNA Panels revealed that the transcript is present in formation of complexes between nuclear proteins and the Sp1 binding site. In an effort to elucidate a wide variety of human adult, fetal and tumor tissues. RT-PCR analysis of the 5Ј end of the the signaling pathway that leads to Dlx3 activation, we have studied the Transforming Growth YEL mRNA in different tissues suggested utilization of two alternate promoters, the most upstream Factor-01β1 (TGF-01β) mediated transcription. Co-transfection experiments with Smad genes, one being exclusively utilized by epidermal keratinocytes. These data suggest that YEL encodes showed that the Dlx3 promoter is activated by the TGF-01β signaling pathway through the a novel transcriptional regulator, which interacts with both DNA, and viral and cellular Smad3 and Smad4 genes. transcription factors. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 637

685 686 Toward Functional Analysis and Identification of the DNA Binding Site of the Hairless Gene Product Genome-Wide Characterization of NF-01κB Target Genes in Human Epithelial Cells Via High V. Aita,* H. Uyttendaele† and A. Christiano*† Density cDNA Microarrays Departments of *Genetics and Development and †Dermatology, Columbia University, New York, K. Hinata, C. Barry, P. Brown and P. Khavari New York VA Palo Alto and Stanford University, Stanford, California Investigations into the relationship between the structure and function of the hairless (hr) gene NF-01κB gene regulatory proteins control important cell fate decisions, have been implicated in provide valuable insight into the specific nature of the transcriptional regulation mediated by this cutaneous inflammation and have recently also been shown necessary for normal epidermal growth gene. Hr is an essential regulator of the hair cycle and has been implicated in a form of congenital control. The identity of target genes mediating NF-01κB effects in skin and other tissues, however, atrichia observed in both humans (OMIM# 209500) and hairless and rhino mice. In humans, is largely unknown. To identify genes regulated by NF-01κB in epithelial cells at a genomic scale, distinct mutations in the h gene have been observed in families from diverse ethnic origins we expressed NF-01κB subunits p50 and p65 in early passage human keratinocytes via high including Pakistani, Irish, Arab Palestinian, Japanese and Polish. Of particular interest, the mutation efficiency retroviral transduction and isolated mRNA for hybridization to cDNA microarrays; in the Polish family is a missense mutation changing the invariant fifth cysteine residue of the replicate cell cultures transduced in parallel for lacZ expression served as controls. Hybridizations zinc-finger binding domain to a glycine. In rhino mice, we have identified four distinct nonsense were performed by both 33P and fluorescence methods on a total of approximately 16,300 arrayed mutations leading to an absense of functional protein, which results in a dysregulation of apoptosis genes. A total of 55 genes demonstrating either strong activation or strong repression by NF- at the first catagen phase of the adult hair cycle and subsequent destruction of the hair follicle. Hr 01κB were identified by this approach for further study. NF-01κB regulated genes were found to has been identified as a putative transcription factor since it contains a single zinc-finger domain. include cell cycle regulators, cytokines, signal transduction molecules and cell adhesion molecules Hr functionally interacts with the thyroid hormone receptor to repress transcription. We demonstrate as well as a number of expressed sequence tag (ESTs) with unknown function. NF-01κB induced that h binds DNA by western blot analysis of proteins captured via their binding to DNA- genes include multiple CDK inhibitors, MCP1, IL-8 and ICAM1. Genes repressed by NF-01κB cepharose beads incubated with total protein lysates from 293T cells overexpressing the h gene. include plectin, laminin 5 01α3 chain, 01α6 integrin, Type XVII collagen, fibronectin and Serial c-terminal deletions and site-directed mutagenesis of the zinc-finger and thyroid hormone connexin 26. Based on these findings and concurrent gene cluster analyses, these data indicate binding domains, as well as a recapitulation of mutations observed in humans, were engineered that NF-01κB appears to induce genes influencing growth arrest and inflammation in epidermis in the h cDNA. They will be utilized in this assay to map the specific structural motifs mediating and to suppress genes expressed in the proliferating cells of the basal cell layer. Consistent with DNA binding activity. Using purified Hr protein, the assay will also determine whether any this, epidermal NF-01κB subunits are found to translocate into the nucleus in cells undergoing cofactors are required for DNA binding. Binding site selection assays such as the target determination physiologic growth arrest within normal epidermis as well in the inflammatory skin diseases assay will elucidate the specific recognition sequence bound by Hr protein, which will be cutaneous T cell lymphoma and lichen planus. These data support a model in which NF-01κB confirmed by gel shift and DNAse protection assays. Development of a functional assay for h promotes a growth arrested state such as that accompanying normal keratinocyte differentiation gene activity will facilitate a better understanding of h gene function and genotype-phenotype as well as the capacity for inducing inflammation, consistent with its activation in inflammatory correlations in humans and mice with congenital atrichia. skin disease and ultraviolet injury.

687 688 c-Jun Mediates UV-Induced Dermal Damage by Coordinately Inducing Collagen Breakdown and Functional Characterization of Keratinocyte-Specific Enhancer Elements in the Human Protein Inhibiting Collagen Synthesis Kinase C h Promoter X. Li, H. Talwar, G. Fisher and J. Voorhees T. Quan and G. Fisher Department of Dermatology, University of Michigan, Ann Arbor, Michigan Department of Dermatology, University of Michigan, Ann Arbor, Michigan Ultraviolet (UV) irradiation impairs dermal collagen by both inducing matrix metalloproteinases Protein kinase C (PKC) 01η is specifically expressed in keratinocytes undergoing terminal (MMP) that degrade collagen and inhibiting procollagen synthesis, in human skin in vivo. Evidence differentiation in human skin. To understand differentiation-dependent, cell-type specific PKC suggests that these deleterious effects of UV on collagen are largely responsible for sun-induced 01η expression, we have cloned and characterized the PKC 01η gene promoter. A 9.4kb fragment premature skin aging. UV induces the transcription factor c-Jun, which activates MMP gene encompassing the 5’-flanking region, first exon, and first intron, was localized on human expression. Jun proteins have also been shown by Mauviel and coworkers [J Bio Chem, 1996, chromosome 14 (14q22–23). Two major transcription initiation sites, identified by RT-PCR and 271(6):3272–8] to inhibit TGF01β-dependent alpha2 type I collagen COL-I(01α2) gene expression primer extension, and confirmed by S1 nuclease mapping, were located approximately 650 bp Therefore, we have examined the role of c-Jun in UV inhibition of COL-I(01α2) expression in upstream from the initiator ATG. Transient transfection assays revealed that 1.6kb 5’-flanking human skin fibroblasts. Fibroblasts were transfected with a reporter plasmid encoding a reporter region displayed maximal promoter activity. The promoter was active in human keratinocytes, gene (CAT) under the control of the COL-I(01α2) promoter. This reporter, like endogenous but not human skin fibroblasts, in accord with endogenous PKC 01η gene expression. Stepwise COL-I(01α2) gene, was constitutively expressed in fibroblasts cultured in serum-containing media. 5’-deletion analysis revealed a keratinocyte-specific enhancer (KSE) region between –1286 bp Exposure of fibroblasts to UV (30 mJ per cm2 from a UVB source filtered to remove UVC) and –1189 bp. The KSE region regulated heterologous promoters in human keratinocytes, but inhibited COL-I(01α2) reporter activity 50% (n ϭ 5). In parallel cultures UV induced c-Jun 3- not human skin fibroblasts. Computer analysis of the KSE region identified a GA-rich region fold (n ϭ 2) and reduced COL-I(01α2) mRNA levels 50% (n ϭ 3). Augmentation of c-Jun containing consensus Ets transcription factor (ETS-TF) and heat shock transcription factor (HS- levels, by cotransfection of a c-Jun expression vector, resulted in 90% inhibition of COL-I(01α2) TF) binding sites. Gel shift, UV cross-linking and DNase I footprint analyses of the KSE region reporter activity by UV. In contrast, transfection of a dominant negative mutant c-Jun expression demonstrated binding of proteins related to ETS-TF and HS-TF family members. Mutations of vector completely prevented UV inhibition of COL-I(01α2) reporter activity. These data indicate the ETS-TF and HS-TF binding sites abrogated DNA-protein complex formation and caused that c-Jun is a key mediator of UV-induced down regulation of COL-I(01α2) gene expression. loss of functional enhancer activity. The above data demonstrate that keratinocyte-specific By both inducing MMPs and inhibiting procollagen synthesis, c-Jun causes a collagen deficiency expression of PKC 01η results from combinatorial effects of multiple cis-acting elements including in UV-irradiated skin, and thereby promotes photoaging. ETS-TF and HS-TF that act in concert with the PKC 01η promoter.

689 690 Cloning and Preliminary Characterization of the Mouse Collagen Type VII Promoter Transplantation of Factor VIII-Expressing Transgenic Skin Corrects Factor VIII Deficiency in a M. Naso, J. Klement and J. Uitto Mouse Model for Hemophilia A Department of Dermatology and Cutaneous Biology, and the Institute for Molecular Medicine, S. Fakharzadeh, Y. Zhang, R. Sarkar and H. Kazazian, Jr Thomas Medical College, Philadelphia, Pennsylvania Departments of Dermatology and Genetics, University of Pennsylvania School of Medicine, Type VII collagen is a member of the collagen superfamily of extracellular matrix molecules which Philadelphia, Pennsylvania localizes primarily in the basement membrane zone of specialized squamous epithelia. In the skin, The epidermis is an attractive tissue for targeting gene therapy for certain genetic diseases, such it serves as a major component of anchoring fibrils, which play a significant role in regulating the as the hemophilias, because it is highly accessible and proteins synthesized and secreted by stability of the attachment of the epithelial basement membrane zone to the underlying papillary keratinocytes can access the systemic circulation. We previously reported correction of factor VIII dermis. Perturbations in type VII collagen synthesis or structure can give rise to dystrophic forms deficiency in a mouse model for hemophilia A by targeting factor VIII expression to the epidermis of epidermolyis bullosa. In an attempt to better understand the regulation of this gene in vivo,we and to a minor degree other stratified squamous epithelia. We generated transgenic mice that have isolated approximately 7 kb of the 5Ј flanking region of the mouse type VII collagen. express human factor VIII under control of the involucrin promoter. Transgenic mice were mated Sequence analysis has revealed regions of high homology with the human promoter, including with factor VIII null mice to yield mice that express factor VIII in the epidermis but not at G-C rich regions involved in the regulated expression of the human gene. Promoter activity was normal sites of its expression. Some factor VIII expression was detected at other sites lined by detected in mouse primary dermal fibroblasts after transfection of constructs containing –432 and – stratified squamous epithelium, such as upper GI and GU structures. Nonetheless, mice from five 2132 relative to the transcription start site of the mouse promoter fused to a chloramphenicol transgenic lines showed both phenotype correction and plasma factor VIII activity. acetyltransferase reporter gene. These data provide insight into the control of mouse collagen VII We now extend these findings by demonstrating factor VIII replacement derived exclusively from expression, and will aid in future studies aimed at understanding its expression in various a focal cutaneous source of factor VIII. Skin explants from transgenic mice were grafted onto developmental and pathological states in vitro and in vivo. immunodeficient factor VIII/RAG-1 double knockout. Two informative graft recipients were obtained. These mice maintained grafts that represented approximately 10%–15% of body surface area. Circulating factor VIII activity was detectable in both mice 5–6 wk after grafting using in vitro assays for plasma factor VIII activity and whole blood clotting. Factor VIII activity levels of about 10% and 5% compared to normal mice were observed. Whole blood from each mouse clotted readily within 15–20 min, whereas whole blood samples from factor VIII deficient control mice did not clot even after several hours. These results provide further evidence that the epidermis may serve as a suitable target tissue for factor VIII gene therapy. 638 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

691 692 Point Mutations in the 1A Domain (N114) of Hair Keratin hHb6 in Monilethrix Patients: A Towards the New Chromosome Locus in Autosomal Recessive Congenital Ichthyosis New Hotspot for Mutations? E. Laiho,* K-M. Niemi,† J. Ignatius,‡ U. Saarialho-Kere,† J. Kere§ and A. Palotie*ϩ B. Korge, H. Hamm,* H. Traupe,† A. Irvine,‡ E. Healy,§ M. Birch-Machin,§ J. Rees,§ A. *Laboratory Department of Helsinki University Central Hospital, Helsinki, Finland, †Department Messenger,ϩ S. Holmes,** C. Jury†† and C. Munro†† of Dermatology, Helsinki University Central Hospital, Helsinki, Finland, ‡Department of Clinical Departments of Dermatology and Venerol., University of Ko¨ln, *Wu¨ rzburg and †Mu¨nster, Neurophysiology, Jorvi Hospital, Espoo, Finland, §Finnish Genome Center, University of Helsinki, Germany; and Departments of Dermatology, ‡Royal Victoria Hospital Belfast, §University of Finland, ϩDepartment of Human Genetics, UCLA School of Medicine, Los Angeles, California Newcastle upon Tyne, ϩRoyal Hallamshire Hosp. Sheffield, and **Royal Infirmary and Autosomal Recessive Congenital Ichthyosis (ARCI) is a heterogeneous group of inherited disorders ††Southern General Hospital Glasgow, UK of cornification. One gene, Transglutaminase 1 (Keratinocyte Transglutaminase) in chromosome Monilethrix (Mt) is an autosomal dominant disorder causing beaded and fragile hair. Variable 14 has been identified as a defective gene in the Lamellar Ichtyosis, a severe scaling disorder alopecia usually presents in infancy, sometimes with follicular hyperkeratosis and nail dystrophy. belonging to ARCI. An additional locus on chromosome 2q33–35 has also been detected, but In most families with Mt studied by linkage analysis, the defect has been localised to the basic the gene is yet unknown. There is also evidence of at least one additional but so far undefined keratin gene cluster on 12q13. Meanwhile several basic human hair keratin genes have been ARCI locus. The objective of our study is to search for new loci and genes for ARCI to further cloned and defects causing Mt have been found in the 2B region of the upper hair cortex hair study the pathogenesis of the scaling disorders. We are searching for a new ARCI locus in one keratins hHb1 and hHb6. To confirm and extend these findings, we have used direct DNA large Finnish ARCI pedigree using the homozygosity mapping technique. The family originates sequencing of PCR amplification products to study the entire rod domain of hHb1 and hHb6 in from a genetically isolated island on the Western coast of Finland. The family contains five affected 18 families with Mt. We have found the same heterozygous lysine substitution of a highly patients born as collodion babies but demonstrated a relatively mild ichthyosis later in life. Linkage conserved glutamic acid residue (Glu413Lys) in the helix termination peptide of hHb6 in three analysis of candidate loci such as TGM 1, 2q33–35 region, Keratin loci on 12q11–13 and 17q, German and three British families. In the hHb1 gene we found the corresponding Glu413Lys Epidermal differentiation complex on 1q21 and Transglutaminase 2 and 3 locus on 20q11.2 have mutation in one British family and another mutation in the nearby residue Glu402Lys in three been excluded as causative loci for ichthyosis in this family. As no linkage to candidate loci has German families and one Irish family. Furthermore, we report for the first time two mutations in been observed, a genome scan using 400 randomly distributed microsatellite markers (modified the 1A domain of hHb6. These novel mutations have been found in British and Portuguese Mt Weber set 8) is performed at present. Our present finding suggests the presence of a third ARCI cases. The same highly conserved residue N114 in the 1A helix initiation peptide of hHb6 was locus and confirms the earlier findings of genetic heterogeneity of ARCI. either mutated to a histidine or an aspartic acid residue. In five other Mt cases mutations in these domains of hHb1 and hHb6 have not been identified. Our findings confirm that defects in cortical basic hair keratins are the molecular basis for most cases of Mt, and suggest that the Glu413 residues in both hHb1 and hHb6, and the Glu402 in hHb1, represent mutational hotspots. Whether N114 represents another mutational hotspot have to be seen.

693 694 Sustainable TGM1 Gene Delivery in Human Lamellar Ichthyosis Patient Skin in vivo Differential Behaviors Toward UVA/B Radiations of Fibroblasts and Keratinocytes from Normal Q. Lin, H. Deng, H. Fan and P. Khavari and DNA-repair Deficient Patients (Xeroderma Pigmentosum, and Trichothiodystrophy) Stanford University, Stanford, California A. Otto, L. Riou, C. Marionnet, A. Sarasin and T. Magnaldo Genetic correction of monogenic human skin disorders represents a potentially effective molecular Molecular Genetics Laboratory, CNRS UPR 42, Villejuif, France therapy for severe diseases for which current therapy is only palliative. Recent gene transfer efforts We have compared the DNA repair capacities (by unscheduled DNA synthesis, UDS) and cell to epidermis have restored normal gene expression to patient skin regenerated on immune deficient survival (by clonal analysis) toward UVA and UVB irradiations of epidermal keratinocytes mice, including transglutaminase 1 (TGase1) in lamellar ichthyosis (LI), steroid sulfatase in X- and dermal fibroblasts grown from normal, xeroderma pigmentosum (XP-C, XP-D) and linked ichthyosis and Type XVII collagen/BP180/BPAG2 in junctional epidermolysis bullosa. trichothiodystrophy (TTD) individuals. XP and TTD are rare (01dA6d5.10–5) genodermatoses While these efforts achieved disease correction at histologic, molecular and functional levels, the (recessive, autosomal) with reduced capacities to repair ultraviolet (UV) induced DNA-lesions duration of such gene delivery failed to extend beyond the approximately 4 wk epidermal turnover [cyclobutane pyrimidine dimers, CPDs; pyrimidine (6–4) pyrimidone 6–4PP]. Despite XP but cycle. Because sustaining cutaneous gene delivery is a necessary step in bringing cutaneous gene not TTD patients are prone to basal-and squamous-cell carcinomas, no comparative studies of the transfer efforts into the realm of clinical feasibility, we utilized recent refinements in gene transfer XP and TTD phenotypes has been performed to date using epidermal keatinocytes. and retroviral vector design to develop a model of more durable gene delivery for genetic skin In any cell type UDS did not increase after UVA irradiation but hierarchies of DNA-repair disease, using LI as a prototype. First, we generated amphotropic retroviral gene delivery vectors capacities of fibroblasts and keratinocytes could be drawn following UVB irradiation. The same with specific deletions in the U3 region of the 3Ј LTR that we have shown previously to produce dose of UVB (1000 J per m2) induced twice more lesions in fibroblasts than in keratinocytes. more durable delivery of a GFP marker gene in human skin tissue in vivo. Previously characterized UV-survival were always higher in keratinocytes than in fibroblasts. Normal and TTD keratinocytes TGase1-deficient LI patient keratinocytes were transduced with these second generation TGase1 survived better toward UVA and UVB radiations than XP-C and XP-D keratinocytes. XP-C expression vectors as well as lacZ control in vitro and high efficiency TGase1 protein re-expression keratinocytes exhibited exacerbated sensitivity toward UVA radiations. UDS values at UV doses confirmed in vitro. These genetically engineered cells were then grafted to SCID mice to regenerate leading to 50% cell survival indicated that the ratio of DNA-repair capacity to cell survival is human skin [n ϭ 4 mice per vector group]. TGase1 expression was sustained in vivo by this higher in keratinocytes than in fibroblasts. In addition, UVA and UVB irradiations induced a approach out to 12 wk postgrafting, the duration of the experiment; controls failed to demonstrate transition from proliferative to abortive keratinocyte colonies. This transition varied between any detectable expression as expected. These data indicate that more durable gene delivery donors and was in part corellated to their cancer proneness. Altogether these data provide the first spanning multiple epidermal turnover cycles can be achieved in human genetic skin disease tissue evidence of differential behaviors of normal, XP and TTD keratinocytes toward UV radiations. and suggest that this approach may be useful in current cutaneous gene therapy efforts.

695 696 Durable Cutaneous Gene Delivery Via Direct Administration of Adenoviral And Lentiviral Vectors Genetic Immunization by Topical Application of Naked DNA to Normal Skin to Human Skin H. Fan, Q. Lin, G. Morrissey and P. Khavari H. Fan, Q. Lin and P. Khavari VA Palo Alto and Stanford University, Stanford, California VA Palo Alto and Stanford University, Stanford, California Naked DNA vaccines delivered via intradermal and intramuscular injection offer promise in Widespread application of cutaneous gene transfer to the treatment of human disease requires new generating specific immune responses, however, these approaches are burdened by the risks, gene delivery approaches. A variety of vectors are available for cutaneous gene transfer, among discomfort and cost of tissue injection. To develop a noninvasive, inexpensive and accessible which adenovirus and modified HIV lentivirus may be attractive due to their high efficiency approach to administering these vectors, we topically applied naked DNA in aqueous solution to infection of nondividing cells. Key currently needed elements include the ability to (i) directly untreated skin in an approach aimed at capitalizing on the effective immune response generated administer vectors to intact skin and to (ii) sustain expression of the delivered gene out to clinically by skin to small amounts of foreign proteins. Topical application of naked plasmid DNA expression meaningful timepoints. In an effort to develop such an approach we topically applied and directly vectors for both E. coli 01β-galactosidase and hepatitis B surface antigen (HBsAg) to intact murine injected lacZ expressing adenovirus, lentivirus, MMLV-based retrovirus and naked plasmid vectors skin induced specific antibody as well as specific cell-mediated immune responses. Antibody to full thickness human skin grafted on SCID mice after confirming that all vectors were active responses demonstrated an early IgM response followed by development of a predominantly IgG in keratinocytes and fibroblasts in vitro. While topical administration of all of these vectors to intact response which could be re-boosted with additional topical application. Specific antigen-induced human skin tissue failed to achieve detectable gene delivery, intradermal injection with both cellular proliferation was sustained for over 6 mo, with no response detected to irrelevant adenovirus (5 01ϫ 109 infectious virions/graft, total of three grafts) and lentivirus (5 01ϫ 106 per polypeptide control. In the case of HBsAg, these responses were at the same order of magnitude graft, total of six grafts) achieved widespread transgene expression. This finding was in marked as those produced by intramuscular injection of the commercially marketed recombinant HBsAg contrast to the minimal expression achieved with both naked DNA and conventional retrovirus. polypeptide vaccine. Topical gene transfer was dependent on the presence of normal hair follicles. Adenoviral and lentiviral-driven gene expression was seen in both epidermis and dermis. Parallel Mice lacking these failed to retain plasmid vectors or to demonstrate evidence of transgene experiments in mouse skin (C57/BL6, three mice per vector) yielded no detectable expression, expression in skin. Hair bearing mice, in contrast, displayed transgene protein expression that indicating potential species-differences in vector effectiveness. Biopsies at 3, 21, 63, 140 and 390 could be detected in skin tissue extracts prepared from biopsies obtained 48 h after topical d demonstrated transgene expression was sustained in human skin tissue for greater than one year. application of naked HBsAg and chloramphenicol acetyltransferase (CAT) expression plasmids, by These findings identify adenoviral and lentiviral vectors as new candidates for cutaneous gene ELISA and CAT assay, respectively. These data demonstrate induction of specific immune responses delivery and confirm that they are attractive candidates for efforts to achieve durable direct gene via simple application of naked DNA vectors to intact skin and indicate that intact hair follicles transfer to the skin. may be necessary for successful gene transfer in this setting. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 639

697 698 Targeted Gene Conversion Mediated by a RNA-DNA Oligonucleotide Using a Lacz Reporter Acral Melanoma is a Distinct Subtype of Cutaneous Melanoma Characterized by Multiple System Gene Amplifications O. Igoucheva, A. Peritz and K. Yoon B. Bastian, M. Kashani-Sabet, D. Moore II, H. Hamm, E. Bro¨cker, P. LeBoit and D. Pinkel Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Philadelphia, Cancer Genetics and Cutaneous Oncology Programs, Cancer Center, and Departments of Pennsylvania Dermatology and Pathology, University of California San Francisco, San Francisco, California; A unique hybrid oligonucleotide composed of both RNA and DNA was shown to correct a and Department of Dermatology, University of Wu¨rzburg, Germany point mutation in a site-specific and inheritable manner in extrachromosomal and chromosomal Controversy exists over whether the classification of cutaneous melanoma into different clinico- targets. The RNA stretches of the oligonucleotide was hypothesized to facilitate the homologous pathological subtypes is justified. In a recent study using comparative genomic hybridization recombination and a mismatch created between the targeted strand and RNA-DNA oligonucleotide (CGH) we found evidence of genetic heterogeneity of melanoma subtypes (Cancer Res 58:2170– may be then repaired by the DNA repair system. We have developed a shuttle vector system to 5,1998). Here we performed CGH on 15 pairs of primary melanomas that were matched for assay targeted gene correction using the chimeric oligonucleotide. A point mutation was made in patient age and tumor-thickness and diagnosed as either acral lentiginous or superficial spreading a plasmid encoding the E. coli 01β-galactosidase resulting a GLU to LYS substitution at amino melanoma (SSM). Two acral melanomas (AM) were from the toe, 10 from the sole, and three acid 461. This mutation resulted in the loss of enzymatic activity. Two chimeric oligonucleotides were from the foot without further specification. By histology, 12 AM were of the acral lentiginous were designed to (i) restore wild type activity in mutant plasmid, and (ii) knock out activity in type, two were unclassifiable, and one had overlapping features with SSM. The total number of wild type plasmid. The activity of the chimeras was assayed in two systems: (i) transient transfection aberrations was significantly higher in the AM group (average of 11.5 per case, vs 5.6). Most of CHO cells and (ii) incubation with nuclear extract derived from CHO cells. After transfection strikingly, we found high-level amplifications in 15 of 15 AM and only in two of 15 SSM. or incubation, plasmid DNA was isolated and transformed into a 01∆lacZ strain of E. coli. The Amplifications in the AMs mostly involved multiple regions (average 1.8, total of 29), whereas frequency of gene correction was scored by blue/white phenotype of the colonies. Using this the two SSM only had a single amplification each. This difference was highly significant (P ϭ system, we were able to measure the targeted correction frequency. This reporter assay should 10–7). Amplifications detected by CGH were confirmed by fluorescent in situ hybridization (FISH) allow us to determine the activity of RNA-DNA chimeric oligonucleotides in different cell on tissue sections revealing DNA copy numbers of the amplicons ranging from six to over 40 systems. Although a homologous recombination event in episome may differ from chromosome, signals per nucleus. Amplifications frequently involved chromosome 11q13 and 22q12, regions a shuttle system enables us to determine the feasibility of gene targeting in a given cell line and harboring several known oncogenes and growth-factors for melanoma. We demonstrate that acral to compare the frequency of targeting among different cells. melanomas are a distinct subtype of melanoma, characterized by a unique form of genomic instability expressed by frequent high-level amplifications.

699 700 Identification of Three Novel Point Mutations in Keratins K1 and K10 in Epidermolytic Hyperker- Durable Laminin 5 01β3 Gene Delivery Vectors for Gene Therapy of Junctional Epidermolysis atosis Bullosa M. Arin, M. Longley, I. Anton-Lamprecht, W. Ku¨ster, E. Epstein and D. Roop P. Robbins, E. Alvarez-Saavedra, Q. Lin, X-J. Chen and P. Khavari Departments of Cell Biology and Dermatology, Baylor College of Medicine, Houston, Texas; VA Palo Alto and Stanford University, Stanford, California Department of Dermatology, Ruprecht-Karls-Universita¨t, Heidelberg, Germany; TOMESA- Junctional epidermolysis bullosa (JEB) involves separation at the dermal-epidermal junction and Clinic, Bad Salzschlirf, Germany; Department of Dermatology, University of California, San results from mutations in genes that encode vital structural proteins of the basement membrane Francisco, California zone, including Type XVII collagen/BPAG2/BP180 and laminin 5. Recently, we genetically Epidermolytic hyperkeratosis (EHK) is an autosomal dominant disorder of cornification character- engineered Type XVII collagen-deficient primary JEB patient keratinocytes to restore normal ized by a varying degree of erythroderma, blister formation and hyperkeratosis. Point mutations gene expression to JEB patient skin tissue regenerated in vivo on SCID mice. Like other genetic in the genes encoding the suprabasal-specific keratins, K1 and K10 have been identified as the correction efforts in human skin tissue, corrective gene expression was not durable, a result underlying genetic defect. The mutant keratin causes a collapse of the intermediate filament (IF) attributed to vector inactivation or failure to target long-lived stem cell progenitors. Recent network which leads to tonofilament clumping and lysis of the affected keratinocytes. We have refinements in retroviral vector design and keratinocyte gene transfer, however, have permitted identified three novel point mutations in the conserved initiation and termination motifs of more durable delivery of marker genes. To develop a more durable approach to corrective gene keratins K1 and K10 in EHK. In a sporadic case of EHK, a single base pair mutation was identified delivery in another JEB subtype, laminin 5-deficient Herlitz JEB, we generated amphotropic which lead to a nonconservative amino acid substitution at the beginning of the rod domain of retroviral expression vectors for the laminin 5 01β3 chain. These vectors were constructed using K10 (Y14S). This substitution may cause conformational changes in the two-chain coiled-coil a backbone vector containing specific deletions in the U3 region of the 3Ј LTR that we have molecule and impede the next stages of filament assembly. Another sporadic case of EHK was the shown previously permits durable delivery of a GFP marker gene in human skin tissue in vivo. result of a conservative amino acid substitution in the 1A region of K1 (N8T). This asparagine Previously characterized early passage laminin 5 01β3-deficient JEB keratinocytes were transduced residue is located in a region that is involved in molecular overlap interactions between neighboring with these vectors as well as GFP control in vitro. In JEB cells transduced with the laminin 5 01β3 keratin molecules and is thought to disrupt the lateral alignment between heterodimers. In a large vector, high efficiency expression of full length laminin 5 01β3 protein was confirmed in vitro by kindred affected by EHK, a point mutation in the 2B region of K1 (I107T) was identified as the immunofluorescence and immunoblot analysis; GFP control-transduced JEB cells were negative underlying genetic defect. This nonconservative substitution may also affect the conformation of as anticipated. Durable laminin 5 01β3 vectors restored JEB cell morphology and adhesive features the coiled-coil molecule and impede interactions within the overlap region between heterodimers. to normal in vitro. These cells were then used to regenerate human skin on immune deficient All three mutations were associated with a severe disease phenotype thus documenting the role mice. Laminin 5 01β3 gene-transduced tissue demonstrated restoration of laminin 5 01β3 protein of these particular residues in filament assembly and stability. expression at the dermal-epidermal junction in vivo, with durability studies currently underway. These findings provide a basis for future efforts to achieve durable gene delivery in human JEB.

701 702 Wild-Type p53 Induces Apoptosis in Pigmented Melanoma Cell Lines SK-mel-23 and 70W Use of Enhanced Green Fluorescent Protein to Monitor Retroviral-Mediated Human Keratinocyte Without Altelating p21Waf1, Bcl-2 and Bax Expressions Gene Therapy In Vivo T. Yamashita, H. Tonoki, T. Moriuchi, H-Y. Jin and K. Jimbow J. Travers, S. Hurwitz, M. Bierhuizen, G. Wagemaker, M. Marques, Y. Pei and D. Spandau Department of Dermatology, Sapporo Medical University School of Medicine; and Department Departments of Dermatology, Pharmacology and the Herman B Wells Center for Pediatric of Cell Biology, Cancer Institute, Hokkaido University School of Medicine, Sapporo, Japan Research, Indiana University School of Medicine, Indianapolis, Indiana; Institute of Hematology, More than 50% of human cancers contain mutated p53, and re-introduction of wild-type p53 Erasmus University, Rotterdam, The Netherlands can induce apoptosis in cancer cells expressing mutant p53. However, only less than 10% of Because of their availability and potential long life span, keratinocytes have great promise as targets melanomas have been reported to contain mutant p53, and no detailed study has been performed for gene therapy involving both skin as well as for systemic disorders. Improvement of gene about the inducibility of melanoma cell apoptosis by wild-type p53. In order to employ the p53- transfer into keratinocytes will be greatly facilitated by markers which will allow both rapid mediated apoptosis for developping melanoma gene therapy, we analyzed p53 expression of three detection and efficient selection of transduced cells. For these purposes, a recombinant version of pigmented (SK-mel-23, G361 and 70 W) and three nonpigmented (SK-mel-24, SK-mel-118 and the Aequorea victoria green fluorescent protein that is enhanced for high-level expression in TXM-18) melanoma cell lines by yeast functional assay, and examined if apoptosis could be mammalian cells (EGFP) was placed into a replication-deficient retroviral vector. High-titer induced in them by p53-expressing recombinant adenovirus. Among six melanoma cell lines retrovirus was used to transduce both primary cultures of neonatal foreskin-derived human tested, only one cell line 70W expressed mutant p53. Sequence analysis revealed that 258th codon keratinocytes (HK) as well as the immortalized keratinocyte-derived cell line HaCaT. Both cell of p53 in 70W cells was changed from GAA (E) to AAA (K). By infecting the recombinant types stably expressed the EGFP, and this marker allowed rapid sorting of transduced cells by adenovirus expressing wild-type p53, apoptosis was induced in not only 70W but also SK-mel- fluorescence-activated cell sorting. EGFP expression was seen in HaCaT keratinocytes for at least 23 cell lines. In order to further study the molecular basis of p53-mediated apoptosis in SK-mel- 40 passages, and the presence of this construct did not effect cell growth. Transduced populations 23 and 70W, cellular proteins related to p53-mediated apoptosis were analyzed by western blotting. of both HK and HaCaT were grafted into nod/scid mice, resulting in a functional epidermis. p53-inducible cdk inhibitor p21Waf1 was not clearly detectable in both apoptotic (SK-mel-23 and EGFP expression was readily seen in vivo by exposing the xenografts to an ultraviolet light source. 70W) and nonapoptotic (SK-mel-24 and SK-mel-118) groups before and after p53-expressing These studies demonstrate the feasibility of using EGFP as a convenient and rapid marker to adenovirus infection. Anti-apoptotic Bcl-2 was detectable in three of them (SK-mel-23, 70W and monitor keratinocyte gene transfer both in vitro and in vivo. SK-mel-24), while apoptotic Bax protein was detectable in both of the groups but only faintly before and after infection of p53-expressing adenovirus. We suggest that cellular target protein(s) other than p21Waf1, Bcl-2 and Bax is responsible for the induction of p53-mediated apoptosis of melanoma cells, and wild-type p53-expressing adenovirus can provide a basis of future development of apoptosis-mediated melanoma gene therapy in particular to those of pigmented ones. 640 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

703 704 Mutations in the Zinc-Finger Domain of the Human Hairless Gene Underlie Congenital Atrichia Mutation Analysis of Sjo¨gren–Larsson Syndrome W. Ahmad,* K. Nomura,‡ U. Kozlowska,§ A. Kozlowska,§ I. Hashimoto,ϩ S. Jablonska§ and W. Rizzo, G. Carney and Z. Lin A. M. Christiano*† Departments of Pediatrics and Human Genetics, Medical College of Virginia, Virginia Common- *Departments of Dermatology and †Genetics and Development, Columbia University, New York, wealth University, Richmond, Virgina New York; ‡Department of Dermatology, Aomori Prefectural Central Hospital, Aomori, Japan; Sjo¨gren–Larsson syndrome (SLS) is an autosomal recessive disorder characterized by ichthyosis, §Department of Dermatology, Warsaw, Poland; ϩDepartment of Dermatology, Hirosaki University mental retardation and spastic diplegia or tetraplegia. The disease is caused by mutations in the School of Medicine, Hirosaki, Japan gene encoding fatty aldehyde dehydrogenase (FALDH), an enzyme that is necessary for oxidation Congenital atrichia (MIM 209500) is a rare autosomal recessive disorder characterized by complete of long-chain aldehydes arising from fatty alcohol metabolism. To determine the genetic basis for loss of hair shortly after birth. We previously reported the identification of pathogenetic missense SLS, we performed mutation analysis on a large group of SLS probands from throughout the and deletion mutations in the human hairless gene in families with atrichia from Pakistan, Ireland world. All patients were confirmed to have deficient FALDH enzyme activity in cultured skin and Israel. As an extension of these studies, here we studied two families, one from Japan and a fibroblasts. FALDH exons were amplified from genomic DNA by PCR and directly sequenced. second from Poland, with one and two members affected with atrichia, respectively. In the affected Among 57 SLS probands, 41 different mutations were detected. These included 19 missense individuals from both families, hair was absent from the scalp, axillae, pubis and other parts of the mutations, three nonsense mutations, nine deletions, two insertions, two complex deletion/ body, and eyebrows and eyelashes were sparse. Natal hairs were present at birth but began to shed substitutions, and seven splice-site mutations. The missense mutations were distributed throughout within first few months of life and completely disappeared by 6 mo to 1 year of age. Skin biopsy the gene and predominantly led to nonconservative amino acid substitutions. Most deletions samples from the affected members from both the families showed the absence of mature hair involved a single nucleotide, but one inbred Turkish patient had a deletion that spanned 3 kb and follicle structures with the formation of cysts filled with keratinaceous material. Sequence analysis eliminated exon 2. Two mutations (del 1297–8 and 943CϾT) were common among European of the human hairless gene from the affected members from both families revealed the presence patients and three other mutations (a complex 3 bp deletion/21 bp insertion, 551CϾT and of mutations in the highly conserved zinc-finger domain of the gene. In the Polish family, a 682CϾT) were frequent among patients from the Mideast. Four single nucleotide polymorphisms homozygous T-to-G transition at nucleotide position 1864 resulted in a cysteine to glycine amino (three intronic and one exonic silent polymorphism) were found in the FALDH gene. Our results acid substitution at position 622 (C622G), which abolished the fifth cysteine of the six-cysteine indicate that SLS is caused by a variety of mutations, which will provide a basis for DNA diagnosis zinc-finger domain. In the Japanese family, a homozygous C-to-T transition at nucleotide position and understanding the correlation between structural and functional alterations in the enzyme. 1837 converted an arginine residue to a termination codon (R613X). The mutated residues are conserved through 90 million evolutionary years among humans, rhesus monkey, mouse and rat, suggesting that they are critical to the function of the zinc-finger domain. The human hairless gene encodes a putative single zinc-finger transcription factor with restricted expression in brain and skin, which regulates catagen remodeling in the hair cycle.

705 706 Hot Spot Mutations in Keratin 2E Suggest a Correlation Between Genotype and Phenotype in A Novel Autosomal Dominant Ichthyosis Patients with Ichthyosis Bullosa of Siemens J. DiGiovanna, S. Bale and P. Fleckman L. Baden,** Y. Suga,* M. Arin,* G. Scott,‡§ L. Goldsmith,‡ C. Magro,ϩ H. Baden** and Div Dermpharm, Brown Univ, Providence, Rhode Island; Genetic Studies Section, LSB, NIAMS, D. Roop*† NIH, Bethesda, Maryland; Department Medicine (Dermatology), University of Washington, Departments of *Cell Biology and †Dermatology, Baylor College of Medicine, Houston, Texas; Seattle, Washington Departments of ‡Dermatology and §Pathology, University of Rochester Medical School, Rochester, Thirteen members of one family and four unrelated spouses were examined in the late fall in the New York; Depts. of ϩPathology and **Dermatology, Harvard Medical School, Massachusetts mid west. Skin bx was obtained in 16 of the 17. This autosomal dominant disorder had onset in General Hospital, Boston, Massachusetts early childhood. No subject was born with a collodion membrane, blistering or erythroderma. Ichthyosis bullosa of Siemens (IBS) is a rare autosomal dominant disorder of cornification which All affected complained of mild to moderate decreased sweating and heat intolerance; two of results from a collapsed intermediate filament network in the upper spinous and granular layers. seven affected and one of six unaffected related had a hx of ‘‘heatstroke’’ in childhood. A strong This is caused by clumping of keratin filaments due to a mutation in the keratin 2e (K2e) gene, hx of atopy was present in three of seven affected and one of six unaffected related subjects. All which inhibits the assembly of higher order structures. The purpose of this study was to determine affected had hyperkeratosis and hyperlinearity of the palms and soles. Chafing, fissuring, and if the differences observed in phenotypic expression can be correlated with the nature of the cracking was present in several, predominantly on the soles. KOH was negative in all affected amino acid substitution, which has not been established in most keratin diseases. Family 1 had tested. The heels displayed prominent fissuring and hyperkeratotsis. A distinctive feature was blisters from birth with more severe clinical involvement, while family 2 had a delayed onset and striking accentuation of the skin markings, with increased skin lines highlighted by powdery, a less severe phenotype. Histopathology showed more severe cytolysis in the granular layer in white scale. The lines were seen circumferentially around the ankles, overlying the achilles tendon, family 1 than in family 2. Sequence analysis was performed on a PCR product corresponding to and extending onto the dorsa of the feet and to the toes. Similar involvement was seen in the a region within the 2B segment of K2e, previously identified as a hot spot for mutation in IBS. web space between the thumb and index finger, extending in some on to the dorsa of the hands In family 1, there wasaGtoAtransition at nucleotide position 1510 resulting in a glutamic acid from the wrists to the fingertips. Thickening of the hyponychium of the fingers and toes was seen to lysine substitution in amino acid residue 117 of the 2B segment. In family 2, there wasaGto in two of seven. Trace to mild erythema of the face, extremities, and occasionally the trunk was T transversion at nucleotide position 1512 resulting in a glutamic acid to aspartic acid substitution, present in six of seven. In most, fine, white scaling was generalized with accentuation on the also at amino acid residue 117 of the 2B segment. Allele-specific PCR analysis confirmed that dorsa of the hands, the knees, distal legs and ankles. However, thin, brown, lamellar-like these changes were only observed in affected family members and not in unaffected individuals. accumulations of scale were seen on the legs in two of seven. Histology showed a prominent The change in the length of the side chain and charge of the nonconservative mutation would granular layer and thickened, compacted stratum corneum. The clinical and histologic finding be expected to have a more dramatic effect on filament assembly than the slight change in length suggest this represents a unique, autosomal dominant ichthyosis, distinct from ichthyosis vulgaris. of the side chain associated with the conservative substitution. Thus, our results suggest that a clear correlation exists between genotype and phenotype in these IBS families.

707 708 Linkage Disequilibrium Mapping of Psoriasis Susceptibility Locus PSORS1 on Chromosome 6 Durable and Efficient Corrective Keratin 14 Gene Therapy in Recessive Epidermolysis Bullosa R. Nair, P. Stuart, T. Henseler, S. Jenisch, N. Chia, E. Westphal, J. Arndt, J. Epperson, E. Simplex (EBS) Christophers, J. Voorhees and J. Elder P. Pereira, L. Bruckner-Tuderman, B. Zabel and M. P. Marinkovich. Department of Dermatology, University of Michigan, Ann Arbor, Michigan; and Departments of University of Muenster and University of Mainz, Germany; Stanford University, Palo Alto, Cali- Dermatology and Immunology, University of Kiel, Germany fornia Genetic analysis by several laboratories including ours [Nair et al. Hum Mol Genet, 1997] has EBS usually arises as a consequence of keratin gene mutations, recessive inheritance occurs in a conclusively established the presence of a susceptibility locus for psoriasis (PSORS1) in the HLA subset of EBS patients and no specific therapy is available. The goal of these studies was to develop region on chromosome 6p21.3. The markers showing evidence of linkage span several megabases a corrective gene therapy for recessive EBS and to develop an animal model with which to study encompassing the HLA complex. Here we report progress in narrowing this interval by linkage EBS. A human keratin 14 (K14) cDNA was generated by RT-PCR from keratinocyte (KC) disequilibrium analysis. The study cohort consisted of 289 independent parent-offspring trios in RNA, verified by sequencing, and ligated into a modified Lazarus retroviral vector containing a which the child has psoriasis. Genotype data was generated for 60 closely spaced microsatellite blasticidin resistance gene for transient selection. Next, primary KC were cultured from the skin markers, including 51 newly developed ones, encompassing the HLA complex. Analysis of the of a recessive EBS patient with absent K14 expression due to K14 gene premature termination data using the transmission/disequilibrium test (TDT) yielded p-values ranging from 0.02 to 10– codon mutations. EBS KC were transduced with K14 cDNA, selected with blasticidin, and then 11 across the entire HLA region extending from HLA-DP to HLA-H (~3.3 Mb) suggesting analyzed. Immunoblots of uncorrected EBS KC lysates using a specific K14 antibody showed extensive linkage disequilibrium in this region. However, the best p-values were obtained for absent expression of K14. In constrast, corrected EBS KC lysates demonstrated a 50-kDa K14 markers in a ~335 kb region between markers TNFB and M6S178. This region contains HLA- band at the same electrophoretic position and intensity as that obtained from normal KC. IF B and -C as well as several other genes. Even in this narrow region, closely spaced markers microscopy using a K14 specific antibody showed absent K14 expression of uncorrected EBS KC, showed substantial variation in the magnitude of the TDT p-values. This variation could not but 100% of corrected EBS KC demonstrated postive K14 expression. Confocal microscopy always be attributed to the polymorphic information content of the marker. Our results demonstrate localized K14 to intermediate filaments in a pattern identical to that of normal KC. In vivo mouse that, at least in the HLA region, TDT analysis of single markers will not be a useful technique to grafting studies of these cells are currently in progress. Next we transferred K14 cDNA to EBS narrow the susceptibility gene interval to less than several hundred kb. Further narrowing of the KC immortalized with HPV E6/7 genes. 100% of immortalized EBS KC showed postitive K14 critical susceptibility region will require construction and detailed analysis of dense multimarker expression for over 10 passages after selection. These studies demonstrate a method of long-term haplotypes. corrective therapy that would be applicable to a subset of EBS patients. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 641

709 710 Ras Inhibition Leads to Epidermal Proliferation In Vivo Effect of Alpha Hydroxy Acids on Cell Signalling Kinases in Dermal Fiboblasts H. Deng, C. Seitz and P. Khavari T. Januario, U. Santhanam Stanford University, Stanford, California Unilever Research, Edgewater, New Jersey Cells in stratified epithelium undergo growth arrest prior to commitment to terminal differentiation, We have previously shown that AHAs such as glycolic and lactic acids can stimulate cell a process that is disrupted in neoplasia. Ras GTPases have been associated with induction of proliferation in vitro. In order to understand the mechanism by which they induce proliferation, epidermal hyperplasia and cancer, however, in normal epidermis Ras proteins are found in growth the effect of AHAs on signalling kinases ERK2, JNK and p38 were examined. These kinases have arrested suprabasal layers. This finding, combined with the fact that Ras can induce permanent been implicated in response to a variety of environmental stimuli. Treatment of dermal fibroblasts growth arrest under certain settings, raises the possibility that Ras function may be important in with lactic or glycolic acid led to a dose dependent decrease in intracellular pH. In addition, normal negative epidermal growth regulation. To test this possibility, we targeted expression of activation of ERK 1/2, JNK and p38 by serum was down regulated by pretreatment of cells with N17 mutant dominant negative H-Ras to the epidermis of transgenic mice using both keratin 14 lactic acid. This implies the involvement of signalling pathways using these kinases in the response [K14] and modified keratin 1 [HK1] promoters after confirming that this N17 mutant blocked of the cells to lactic acid and this response is likely triggered by transient intracellular acidification. keratinocyte Ras signaling in vitro. Multiple founders and four stable independent lines of N17 transgenic mice were generated; keratin promoter-targeted constitutively active V12 mutant ras mice were also generated as gain-of-function controls. K14-N17, HK1-N17 and K14-N17 X HK1-N17 double transgenic mice display evidence of blockade of endogenous Ras signal transduction, as measured by inhibition of MAP kinase phosphorylation in vivo. This Ras inhibition appears transgene gene dose-dependent and is associated with massive epidermal hyperplasia in vivo, without alteration of differentiation marker gene expression. N17 epidermis exhibits Ͼ5-fold increase in the BrdU labeling index in vivo without an increase in TUNEL positive cells, indicating that this hyperplasia is due to increased cellular proliferation rather than inhibited cell death. Clinically, N17 mice display widespread hyperkeratosis and cutaneous erythema with superficial erosions and develop corneal scarring leading to blindness. In addition, N17 transgenic mice fail to complete bone formation in distal limbs and digits, with the most severely affected mice failing to develop hind legs. These data suggest that Ras promotes growth arrest in normal stratified epithelium and that Ras-dependent epidermal signaling may play an important role normal mammalian skeletal development.

711 712 Withdrawn The Role of Notch4 in Endothelial Cell Development H. Uyttendaele, J. Ho,* A. Christiano, J. Rossant* and J. Kitajewski† Departments of Dermatology and †Pathology, Columbia University, New York, New York and *Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada Evidence for involvement of Notch genes in vascular disorders has come from analysis of the human hereditary condition CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy). This disorder is manifested by stroke and dementia and characterized by nonamyloid and nonatherogenic angiopathy of cerebral arteries. The CADASIL locus maps to the human Notch3 gene and mutations in Notch3 have been defined in CADASIL patients. Further evidence for a role of Notch (a transmembrane receptor) in vascular development is suggested by analysis of mice with targeted disruption of genes encoding Notch ligands (Delta like-1 and Jagged-1) which develop severe hemorrhages. To study how alterations in Notch signaling may disrupt vascular development, we have used in vitro and in vivo model systems to analyze the role of Notch4 during vascular development. Rat brain micro vessel endothelial cells (RBE4) were used as a model system to study the role of Notch4 and Jagged-1 in endothelial cell differentiation. Both Notch4/int-3 (a constitutive activated Notch4 allele) and Jagged-1 were able to induce cellular structures with morphology and biochemical properties of endothelial micro vessels. To study the in vivo role of Notch4 in endothelial cell development, homologous recombination in embryonic stem (ES) cells was used to drive expression of Notch4/int-3 from the Flk-1 promoter. This conferred endothelial cell specific expression of Notch4/int-3 starting at day 7 p.c. Mouse embryos that express Notch4/int-3 in the endothelium die at day 10 p.c. and display enlarged and collapsed blood vessels prior to death. Embryoid bodies (EB) were generated from the ES cell lines to investigate the development of the endothelium. Vascular networks observed in EB that express Notch4/int-3 are disorganized and do not form the fine and homogenous networks. These data thus suggest that Notch4/int-3 can disrupt vascular remodeling and is required for proper endothelial cell development.

713 714 IL-8 Receptor Expression Occurs in Normal Epidermal Regeneration and Is Highest in Psoriasis Specific Role of TGF-01β 2 during Hair Follicle Development Skin Equivalents K. Foitzik, R. Paus,* T. Doetschman† and G. P. Dotto N. Konstantinova, D. Woodley and M. Duvic Cutaneous Biology Research Center, Harvard University, Boston, Massachusetts; *Department of Nioxin Research Laboratories, Atlanta, Georgia; North-western University, Chicago, Illinois, and Dermatology, Charite´, Humboldt-Universita¨t zu Berlin, Germany; †Departments of Molecular Dermatology, Maryland Anderson Cancer Center, Houston, Texas Genetics, Biochemistry and Microbiology, University of Cincinnati, Cincinnati IL-8 stimulates keratinocyte proliferation, attracts neutrophils, and may participate in the pathogen- Members of the BMP/TGF-01β family of growth factors are potent regulators of cell growth esis of psoriasis. Two IL-8 Receptors (IL-8R01α, CXCR-1) and (IL-8R01β, CXCR-2) are and differentiation. TGF-01β isoforms show distinct expression patterns during hair follicle members of transmembrane domain rhodopsin superfamily and function in signal transduction. morphogenesis, suggesting important isoform-specific functions during development, but their IL-8Rs are expressed in neutrophils, and monocytes. IL-8R01α, expressed on T cells, has high exact roles remain unclear. Here, we assess the contribution of the individual TGF-01β isoforms specificity for IL-8, while IL-8R01β binds many other members of the CXC chemokines. To to murine hair follicle development. study the role of intrinsic skin cytokines in the pathogenesis of psoriasis and in wound healing, Newborn skin with hair follicles in all stages of hair follicle development serves as a model to we established skin equivalents (SE) with five psoriasis lesional fibroblast lines (PsL), five adult dissect epithelial–mesenchymal interactions during organogenesis. We analyzed histologically the normal fibroblast lines (AdNL), and eyelid fibroblast lines (EyeNL) combined with neonatal developmental stages of hair follicles in newborn TGF-01β 1, 2 and 3 knock-out mice. In TGFβ2 foreskin keratinocytes. Previous work (J Invest Dermatol 107:615, 1996) showed that PsNL and deficient mice a profound delay in hair follicle morphogenesis with hair follicles mostly in the PsLes SE produced higher IL-8 levels than NLSE, correlated with epidermal thickness and hair peg stage, a reduced number of follicles and a 50% thinner skin could be detected. TGF- invagination. To determine the expression of IL-8 receptors in SE, immunochistochemistry with 01β1 null mice showed a slightly more advanced hair follicle development, while follicle specific antibodies for IL8-R01α/β was performed. During maturation of the epidermis, IL- morphogenesis in TGF-01β3 knock-out mice was not affected. The hypothesis that TGF-01β2 8R01β was strongly expressed at days 7–14 in PsLes, AdNL, and EyeNL SEs in the basal layer induces hair follicle formation was directly tested by evaluating the effects of the added factors on and diffusely in the basement membrane zone. IL-8R01α staining was not detected at 7, 10, or wt fetal hair follicle development using beads. Consistent with the knock-out data, TGF-01β2 14 d in NL SE, but was weakly present at day 7–10 in EyeNL SEs where epidermis invaginated treated skin explants showed more hair follicles than control skin. Our data indicate TGF-01β2 into dermis. At day 14, when epidermis reached maximal thickness and invaginations had flattened, plays a specific function, not shared by TGF-01β1 and 3, as a promoter of hair follicle initiation IL-8R01α was no longer present. In comparison, IL-8R01α was detected in PsSE in dermal and development. fibroblasts and in the epidermal monolayer at day 7 and was present throughout the epidermis by day 10. On day 14, IL-8R01α was only expressed under the stratum corneum and in the basal layer. The expression of IL-8 appears to be correlated with that of its receptors and may form a paracrine loop that is important for epidermal regeneration and for neutrophil recruitment in psoriasis. Psoriasis skin equivalents express higher amounts of both IL-8 and its receptors than normal skin equivalents, supporting the hypothesis that psoriasis skin has an intrinsic regenerative phenotype conferred by fibroblasts, even in the absence of T cell infiltrates. 642 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

715 716 Characterization of Cytokine and Chemokine Cxpression in I01κB01α-Deficient Mice in Relation Soluble Factors of Follicular Dermal Papilla Arrest Cell Cycle and Induce Differentiation in to Onset of Severe Dermatitis Follicular Keratinocytes J. Klement L. Tang, H. Lui, S. Madani, D. I. McLean and J. Shapiro Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Philadelphia, Division of Dermatology, The University of British Columbia, Vancouver, BC, Canada Pennsylvania The hair follicle (HF) is the only organ system that periodically and regularly regenerates itself Transcription factor NF-01κB is composed of a protein dimer involved in signal transduction in throughout life. This cycling process is modulated by both systematic and environmental influences, the immune and other systems. Inactive NF-01κB is cytoplasmically retained by protein–protein but at the core of this process are the interactions between the mesenchymal (follicular dermal interaction with I01κB01α. Upon stimulation by cytokines, UV light, viruses, and other agents, papilla (FDP) and dermal sheath) and epithelial (follicular keratinocytes (FKC) and melanocytes) I01κB01α is rapidly degraded releasing NF-01κB to be translocated to the nucleus resulting in cell populations. Dermal–epidermal interactions have long been studied in embryonic skin gene activation. I01κB01α-deficient mice were derived to study this component in the signal appendage development. The postnatal dermal–epidermal interactions are less clear. To study FKC transduction pathway. Previously, we had identified a 4–5 fold increase in TNF01α mRNA in cell growth and differentiation influenced by FDP cells, the conditioned medium (CM) of the IkBa deficient mice from 5-d-old pups. In addition to TNF01α, strong induction of MIP101α, FDP cell culture from normal persons were added to the FKC cell culture and the cell growth MIP-2, and MCP-1 and weak induction of Eotaxin and GM-CSF were found using RNase rate was determined by colorimetric MTT (tetrazolium) assay. The growth rate of FKC cells protection assays and RNA from skin of affected and nonaffected pups. Samples were analyzed treated with CM exhibited a dose-dependent reduction. The treated FKC cells were more from 18-d-old embryos, 1-d-old pups, and 5-d-old pups and interestingly, only TNFa showed elongated and flattened, and cells were aggregated, indicating that these cells were induced to any increase in mRNA levels prior to day 5 postnatal. Although the embryonic level of TNFa undergo differentiation. Five FDP cell lines were tested and similar results were obtained. Flow was elevated, it was only 1.5 fold over wild-type and heterozygous IkBa embryos. The results cytometry analysis showed no sign of apoptotic cell death. However, the treated cells were arrested currently suggest that NF-kB may be constitutively active in embryonic skin of IkBa deficient at G2 phase of the cell cycle. This was further supported at the molecular level by dephosphorylation mice and this activity results in the abnormal expression of cytokines and chemokines resulting in of the cell cycle kinase CDC2 on Western blotting analysis after the treatment with CM. CDC2 the developing dermatitis. kinase regulates G2/mitosis transition and its dephosphorylation is associated with G2 arrest. Another cell cycle kinase CDK2, which is required for the progression through early stage of the cell cycle, was not changed after CM treatment. The cell cycle inhibitor p27 was dramatically induced in the treated FKC cells. This protein has been shown to be associated with cell cycle arrest and be able to initiate differentiation in various cell types including epidermal keratinocytes. The FDP conditioned medium were also added to the hair follicle organ culture to test its growth arrest effects on hair growth ex vivo. The HF growth rate was reduced from 3.8 Ϯ 0.5 mm in the control medium to only 0.9 Ϯ 0.01 mm in the FDP CM after 12 d in culture. We conclude that cultured FDP cells secret soluble factors to induce FKC growth arrest in vitro and reduce HF growth ex vivo. The cell growth arrest might be an integral part of cell differentiation in the FKC. It is hypothesized that this growth regulatory pathways by FDP cells are linked to the control of differentiation-specific gene expression.

717 718 Normal Human Keratinocytes Require ErbB Tyrosine Kinases Other Than ErbB1/EGFR Interleukin-8 Binds to a G Protein Linked Receptor and Signals Through G01α16 in Keratinocytes for Growth B. McAlpine, G-J. Wu, L. Xin and N. Bunnett S. Stoll and J. Elder Department of Dermatology, Emory University, Atlanta, Georgia; and Department of Surgery, Department of Dermatology, University of Michigan, Ann Arbor, Michigan University of California, San Francisco, California The ErbB family of receptor tyrosine kinases (ErbB1, 2, 3, and 4) interact in a hierarchical fashion Multiple inflammatory mediators have been proposed to play a role in inflammatory skin diseases. to effect signal transduction in response to two major ligand families (EGFs and NDF/heregulins). IL-8 is one such mediator implicated in the pathogenesis of psoriasis. IL-8 is a chemokine that We have previously shown that ErbB receptor tyrosine kinase (TK) activation is an important binds to a G protein linked receptor, which transduces signals of activation through a heterotrimeric early step in wound healing. However, the ErbB-TK inhibitor PD153035 was 10 times less potent G protein. We have previously demonstrated that G01α16 mRNA is present in keratinocytes, as an inhibitor of organ culture gene responses than it was as an inhibitor of EGF-induced TK but not in other constituent cells in the skin including fibroblasts, melanocytes and microvascular activity in A431 cells (which strongly overexpress ErbB1 relative to the other ErbBs). Furthermore, endothelial cells. We hypothesize that IL-8 specifically signals through G01α16 in keratinocytes. organ culture-induced gene expression in ErbB1–/– mice was very similar to that observed in Stable transfections into A431 cells of key sense and antisense Ga16 cDNA segments were prepared wild type animals. These results suggested that other members of the ErbB family play important with lipofectin using the pcDNA 3.1 vector and selection Ga16 with G418. Normal human roles in wound healing. To test this hypothesis, we have used three inhibitors (PD153035, 158780 keratinocytes and transfectants were stimulated with IL-8 and the production of inositol triphosphate and 168832) displaying varying specificity against the different ErbB family members. All three (IP3) measured. IP3 production was increased with IL-8 stimulation in normal human keratinocytes compounds have been shown to inhibit EGF induced tyrosine phosphorylation in A431 cells at and sense transfectants, but not in antisense transfectants. These results support the hypothesis that low nM concentrations (13–24 nM). In contrast, PD158780 and PD153035 inhibit NDF-induced IL-8 is signaling through Ga16 in keratinocytes since no significant increased IP3 production was TK activity in MDAMB453 cells (an assay for ErbB TKs other than ErbB1) at 52 and 195 nM, detected after IL-8 stimulation in antisense G01α16 transfectants where G01α16 production has respectively, while PD168832 displayed an IC50 Ͼ 1000 nM (Biochem Pharmacol 54:877). Normal been interrupted. These findings are significant since heterotrimeric G proteins are potential targets human keratinocytes (KC), plated at 5000 cells per cm2, were grown in modified MCDB153 for pharmacological agents. Blocking IL-8 signal transduction through G01α16 may be useful in medium in the presence or absence of various concentrations of each inhibitor. After3dKC treatment of inflammatory skin diseases such as psoriasis. were trypsinized, counted and cell numbers expressed as percentage of DMSO-treated controls. The two ‘‘pan-ErbB’’ inhibitors, PD153035 and PD158780, were similarly potent in their ability to inhibit KC growth [PD153035 IC50 ϭ 90 nM (n ϭ 5), PD158780 IC50 ϭ 118nM(nϭ3)]. In contrast, the ‘‘ErbB1-specific’’ inhibitor PD168832 displayed an IC50 of 1390 nM (n ϭ 3). These results demonstrate for the first time that members of the ErbB family other than ErbB1 can play an important role in regulating KC growth.

719 720 Proliferation-Dependent Nuclear Localization of S100A2 in Human Keratinocytes RAG2–/–:I01κB01α–/– Chimeras Display Psoriasiform Phenotype J. Fu and J. T. Elder C-L. Chen, F. Yull, N. Cardwell, N. Singh, L. Nanney and L. Kerr Department of Dermatology, University of Michigan, Ann Arbor, Michigan Departments of Microbiology and Immunology, Plastic Surgery, Vanderbilt University, Nash- S100A2 has been suggested to function as a tumor suppressor gene, based on its markedly decreased ville, Tennessee expression in tumor-derived cell lines relative to their normal counterparts. We have found that NF-01κB is a ubiquitous transcription factor involved in the signal transduction pathways regulation CaN19 is expressed in SCC and in psoriatic lesions but not in BCC, and have therefore postulated immune response, inflammation and acute phase reaction. NF-01κB is controlled by inhibitory a role for S100A2 in regenerative epidermal differentiation (Cancer Res 57:3055). Available evidence I01κB proteins, mainly I01κB01α. I01κB01α deficient mice, generated in our lab and others, suggests that epidermal differentiation is regulated by the activation of specific transcription factors exhibit a severe dermatitis with neonatal lethality. In order to study the effect of constitutive NF- (TFs), including members of the basic helix-loop-helix (bHLH) family (EMBO J 14:5646). As 01κB activation on the immune response of adult mice, we utilized RAG2–/–blastocyst other S100 proteins have been shown to alter the DNA-binding properties of bHLH TFs (JBC complementation. I01κB01α–/– ES cells were injected into RAG2–/–blastocysts to generate 272:23930), we have hypothesized that S100 A2 might regulate regenerative keratinocyte (KC) chimeric mice in which all mature B and T cells are derived from the I01κB01α–/– ES cells. differentiation by interacting with TFs in the nuclear compartment. As a first test of this hypothesis, The skin of the face, ears, and footpads of these chimeras displayed an extreme flaky phenotype. S100A2 was localized in normal human KC by immunofluorescence (n ϭ 3 independent donors). Histologic examination of affected regions revealed a greatly thickened, hyperproliferative epidermis NHK were seeded on glass slides at various densities, under either serum-free (Boyce-Ham) or with marked acanthosis and hyperkeratosis, neutrophilic infiltration in the epidermis as well as a serum-containing (Rheinwald-Green) culture conditions. Slides were fixed in 4% paraformal- prominent dermal inflammatory infiltrate. Furthermore, adoptive transfer of lymph node from a dehyde, permeabilized with 0.5% Triton X-100, and stained with either of two mouse anti- chimera with this phenotype to a RAG2–/–mouse was capable of producing this psoriasiform S100A2 mAbs (Sigma or Transduction Labs, 1 mng per ml). In both culture systems, S100A2 phenotype in the recipient after 4 wk. Full-thickness noninvolved skin from affected chimeras localized to the nucleus and cytoplasm of all KC in small colonies (16–32 cells). Nuclear staining and their nonaffected littermates was grafted onto nu/nu mice and maintained for 12 wk. When was lost at the centers of KC larger colonies and in confluent KC monolayers, where KC are the grafts were challenged with mild trauma, those from affected chimeras developed a psoriasiform known to be nonproliferative. Both mAb gave very similar results, and isotype controls were phenotype in both the dermis and epidermis complete with microabscesses (the Koebner negative. Transfection of an epitope-tagged S100A2 construct into a tumorigenic, S100A2- phenomenon) while those grafts from nonaffected littermates displayed an unremarkable transient negative carcinoma-derived cell line yielded exclusively cytoplasmic staining (n ϭ 4 independent wounding response. In summary our in vivo data compiled from three differing circumstances in transfectants). Taken together, these results suggest the hypothesis that S100A2 maintains prolifera- mice suggest that constitutive NF-01κB activation (by elimination of its inhibitory protein, tion in differentiation-competent cells by inhibiting the activity of terminal differentiation-specific I01κB01α) is an attractive strategy for the study of psoriasiform dermatoses. transcription factors VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 643

721 722 Delayed Wound Healing in CXCR2 Knockout Mice Involvement of Multiple Mitogen Activated Protein Kinase (MAPK) Pathways in Growth Factor- R. M. Devalaraja,*§ L. Nanney,*† T. Daniel‡ and A. Richmond*‡ Induced Proteinase Expression and Keratinocyte Migration *Departments of Cell Biology, †Plastic Surgery and ‡Medicine, Vanderbilt University School of L. J. McCawley, E. V. Wattenberg,* S. Li,* M. Benevidez,† J. Halbleib† and L. G. Hudson† Medicine and §Veterans Affairs Medical Center, Nashville, Tennessee Department of Cell Biology, Vanderbilt University, Nashville, Tennessee; *Division of Environ- The process of wound healing is dependent upon cell migration and proliferation, which is mental and Occupational Health, University of Minnesota, Minneapolis, Minnesota; †Program in mediated in part by CXC chemokines. Though MGSA (melanoma growth stimulatory activity)/ Pharmacology and Toxicology, University of New Mexico Health Sciences Center, Albuquerque, GRO (growth regulated protein) was initially cloned for its growth stimulatory functions, later New Mexico in vitro and in vivo studies showed its chemotactic and angiogenic properties. The immunomodula- The epidermal growth factor (EGF) receptor is frequently overexpressed in squamous cell tory activity of MGSA/GRO in recruiting neutrophils, lymphocytes and macrophages to the site carcinoma (SCC). The functional consequences of receptor overexpression are not fully understood; of inflammation following wounding has been well established. In our laboratory, we previously however, we have found previously that elevation of EGF receptor expression in human SCC demonstrated that MGSA/GRO and its receptor CXCR2 are expressed in keratinocytes of burn cell lines enhances ligand-dependent migration and proteinase expression. In this study, we wounds at areas where re-epithelialization occurs, implicating MGSA/GRO in the pathophysiology investigated the contributions of EGF-induced MAPK signal transduction cascades to proteinase of wound healing. The receptor for the murine homologs of MGSA/GRO, MIP-2 and KC is expression and migratory responses. Stimulation of SCC 12F cells with EGF promotes colony murine CXCR2. To further examine the significance of CXCR2 binding chemokines in wound dispersion, matrix metalloproteinase (MMP)-9 induction and in vitro invasion. EGF also activates healing, we obtained CXCR2 knock-out mice generated by gene targeting (Cacalano, G et al. the p42/44 extracellular signal regulated kinase (ERK), jun N-terminal kinase (JNK) and p38/ Science, 265:682–684, 1994). Full excision punch wounds or superficial tail stripping wounds were HOG MAPK signaling cascades. Inhibition of MEK-1 by PD98059 inhibits growth factor-induced inflicted on CXCR2 homozygous (–/–), heterozygous (–/ϩ) or wild type (ϩ/ϩ) mice. Mice p42/44 ERK activation and MMP-9 induction. It has been reported that EGF-dependent JNK were sacrificed and wound healing parameters were histologically analyzed at day 3, 5, 10 and 14 activation requires phosphatidylinositol 3-kinase (PI3K) activity and we observe disruption of postwounding. Quantitative analysis of neutrophilic infiltration as well as epithelial maturity EGF-stimulated MMP-9 induction in the presence of PI3K inhibitors thereby suggesting suggested a significant delay in wound repair in mice deficient in CXCR2. In ϩ/ϩ mice, wounds involvement of JNK in the response. In addition, inhibition of p38/HOG also impairs MMP-9 were typically resurfaced at days 3–4 whereas it took almost 14 days to achieve 100% resurfacing induction by EGF. When submaximal concentrations of inhibitors are administered in combination, in CXCR2 knockout mice. In vitro wound healing experiments performed with keratinocytes inhibition of EGF-dependent response is greater than that observed with any single agent. These obtained from two day-old pups of the three different CXCR2 genotypes (–/–, –/ϩ, ϩ/ϩ) findings demonstrate that EGF concurrently activates multiple MAPK signaling cascades and concurred with our in vivo epithelial data. These studies clearly implicate the pathophysiological suggests that coordinate regulation of each pathway is required for maximal EGF-dependent effects of CXCR2 binding chemokines in the process of wound healing. migration and proteinase induction.

723 724 Analysis of the up T Cell Immune Response to Murine PDV Squamous Cell Carcinoma Effect of Vaccine Therapy on the Presence of Melanoma Cells in the Circulation M. Girardi, R. Tigelaar, R. Filler and A. Hayday* J-C. Bystryn, J. Albrecht, R. Oratz, R. L. Shapiro, D. L. Chen, M. C. Rivas and A. Conrad Department of Dermatology, Yale University School of Medicine, New Haven, Conneticut, and Departments of Dermatology, Medicine and Surgery, NYU School of Medicine, New York, New *Department of Immunobiology, University of London, UK York and National Genetics Institute, Santa Monica, California The increased incidence of squamous cell carcinoma (SCC) in renal transplant and other Melanoma cells are present in the circulation of pts with melanoma. Thus, changes in their immunosuppressed patients strongly suggests that the immune system plays a role in protection number may provide an early indication of the effectiveness of therapeutic interventions against SCC development. PDVC57, a keratinocyte tumor cell line that grows as an adherent To assess this possibility, we first examined how often circulating melanoma cells could be detected squamoid population in vitro, rarely results in tumor formation when injected intradermally (i.d.) in 70 pts with melanoma, and whether their presence was related to the extent of the tumor. into syngeneic immunocompetent C57BL/6 (B6) mice. These tumors demonstrate the histologic PCR was used to detect melanoma cells that expressed the melanoma associated antigens MART- findings of SCC. To begin to study the T cell antitumor response against SCC in vivo, we injected 1, MAGE-3, tyrosinase and/or gp100. Melanoma cells that expressed one or more of these groups of B6 and syngeneic T cell receptor (TCR) 01β,01δ, and 01βϫδ knockout mice (01β–/– markers were detected in 23 (33%) of the pts. The cells were present more often in advanced ,01δ–/–, and 01βϫδ–/–) with 1 01ϫ 106 PDVC57 cells i.d. (two sites per mouse). Only 16.7% disease, i.e. in 15 (28%) of 54 pts with stage IIB or III disease versus in eight (50%) of 16 pts with (five of 30) of B6 and 30% (three of 10) of 01δ–/– sites injected developed into tumors, as opposed stage IV disease. We then assessed whether circulating melanoma cells were affected by treatment to 100% (10 of 10) of 01β–/– and 100% (10 of 10) 01βϫδ–/– sites, suggesting an antitumor role with a polyvalent melanoma vaccine; by measuring these cells at baseline and following 2 and 4 of 01αβ T cells in this model. Furthermore, the tumors that did develop in mice with 01αβ T mo of vaccine therapy in 36 of these pts. The vaccine, prepared from shed antigens, contained cells (B6 and 01δ–/–) so at a delayed onset (5–6 wk) than mice lacking 01αβ T cells (01β–/– and multiple melanoma antigens including MART-1, MAGE-3, tyrosinase and gp100. Immunizations 01βϫδ–/–; 2–3 wk). Anti-tumor immune response were long-lived; i.e. all B6 mice demonstrating were given q2–3 weeks x4 and then monthly 01ϫ 3. Prior to vaccine treatment, circulating immunoprotection on initial challenge were resistant to rechallenge up to 3 mo later. Immunopro- melanoma cells were detected in 13 (36%) of the pts. Following 2–4 mo of vaccine treatment, tection was adoptively transferred to 01β–/– mice using as few as 1 01ϫ 106 splenocytes from melanoma cells that expressed any of the markers were no longer detectable in 10 (77%) of the immunoprotected B6 mice (five of six), where as 10 01ϫ 106 splenocytes from naive B6 mice previously positive 13 pts. afforded no protection. New tumor cell lines were established by in vitro culture of trypsinized These observations suggest that the presence of melanoma cells in the circulation is related to the tumors isolated from PDVC57 injected B6 and TCR knockout mice. These lines demonstrate extent of the melanoma, and that their disappearance may provide an early marker of the efficacy morphological differences in vitro, suggesting expression variability. Representational difference of vaccine or other therapies. Ultimately, the utility of the PCR assay will need to be confirmed analysis will be utilized to determine which genes are up- or down-regulated during tumor through clinical follow-up of each patient. development under the various T cell backgrounds. Studies involving the utilization of T cell deficient mice in this model should help in our understanding of immune responses to SCC, and may have implications for tumor immunology in general.

725 726 A Chimeric Mouse Model for Functional Studies of Human Endothelial Cells Differential Requirement for 01γ/01δ T cells in Epicutaneous Versus Airway Sensitization to J. Schechner, A. Nath, C. Hughes, M. Sierra-Honigmann, M. Kluger and J. Pober Soluble Protein Depts. of Dermatology and Pathology, Yale University School of Medicine, New Haven, Con- C. Herrick, R. Tigelaar and K. Bottomly neticut Department of Dermatology and Section of Immunobiology. Yale University School of Medicine, A chimeric human-immunodeficient mouse model has been developed which allows the study New Haven, Conneticut of human endothelial cell (EC) function in vivo. Cultured human dermal microvascular EC The role of 01γ/01δ T cells in generation of Th2 immune responses following exposure to (HDMEC) and human umbilical vein EC (HUVEC) were suspended in a 3-dimensional collagen- ovalbumin (OVA) either epicutaneously (e.c.) or through the airway was investigated. C57BL/6 fibronectin matrix, and allowed to form vessel like tubes in vitro. These vascular constructs were wildtype (WT) or 01γ/01δ T cell deficient (01γ/01δ–/–) mice were initially exposed to OVA implanted subcutaneously into SCID-beige mice. After a 14–21-d period during which the human either e.c. (100 01µg under an occlusive patch) or intranasally (i.n.) (100 01µg daily 01ϫ 3). Two vessels anastomosed with the mouse circulation, the vascular constructs were harvested for weeks later, mice were repeatedly challenged with OVA (25 01µg) i.n. over a 6-d period. Airway histologic examination. Constructs containing HUVEC were implanted into 15 mice. Human inflammatory responses were assessed by analysis of bronchoalveolar lavage (BAL) fluid composition. vascular profiles (detected with UEA-1 lectin) containing mouse erythrocytes, indicative of Compared to sham-immunized mice, increased numbers of cells were recovered from BAL of perfusion, were detected in 14 of these mice. Perfused human vascular profiles were also detected mice sensitized to OVA either e.c (7.9 Ϯ 0.9 vs 1.7 Ϯ 0.2 01ϫ 105) or i.n. (7.5 Ϯ 1.8 vs 1.7 Ϯ in eight of the 10 mice which received HDMEC containing vascular constructs. To assess the 0.6 01ϫ 105) with a high percentage of eosinophils (54% vs Ͻ1% for e.c.; 24% vs 1.5% for i.n.) utility of this model in studying lymphocyte–endothelial interactions, an additional 16 mice in the indicating generation of Th2 type immune responses. When 01γ/01δ–/– mice were exposed to HUVEC experiments received i.p. injections of human PBMCs allogeneic to the EC. Three each OVA, only mice sensitized e.c. showed airway inflammation equivalent to WT controls (6.8 Ϯ of the PBMC recipient and no-PBMC control mice were sacrificed 3 d after PBMC inoculation. 1.9 vs 9.1 Ϯ 1.5 01ϫ 105 total cells; 4.4 Ϯ 1.5 vs 5.9 Ϯ 1.4 01ϫ 105 eosinophils). OVA-specific HLA-DR was upregulated on the endothelium of all the PBMC recipient mice, with no detectable IgG1 antibody responses were also similar in e.c. sensitized WT and 01γ/01δ–/– mice (119,860 expression in the controls, indicating endothelial activation by the PBMCs. By 10 d there was Ϯ 58,940 vs 78,310 Ϯ 13,100 ng per ml). In contrast 01γ/δ– /– mice sensitized by i.n.OVA also a significant decrease in the density of perfused vascular profiles per 10401µ2 in the PBMC exposure had decreased total BAL cell numbers as compared to WT mice (1.9 Ϯ 0.2 vs 7.5 Ϯ recipient mice (2.6 01Ϯ 0.99) compared to the controls (12.4 01Ϯ 2.77, P ϭ 0.002). This loss 1.8 01ϫ 105; P ϭ 0.01) and decreased numbers of eosinophils (0.2 Ϯ 0.1 vs 2.2 Ϯ 1; P ϭ 0.04) of vasculature was presumably due to immune-mediated apoptosis. These data show that HUVEC following airway OVA challenge. However, airway sensitized 01γ/01δ–/– mice did have similar and HDMEC vascular constructs implanted into mice survive to anastomose with, and be perfused levels of anti-OVA IgG1 in serum as compared to WT mice (12,920 Ϯ 4940 vs 6980 Ϯ 2730 ng by, the mouse circulation. There was evidence that these vascular constructs can interact with per ml). Thus, while 01γ/01δ T cells are critical for generation of Th2 type immune responses human PBMCs, suggesting that this model will be useful for studying the interaction of human following airway sensitization, effective Th2 sensitization to OVA can occur independently of lymphocytes and endothelium in vivo. 01γ/01δ cells through the skin. 644 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

727 728 α β α Characteristics of Induced Alopecia Areata-Like Hair Loss in C3H/HeJ Mice Are Consistent with Integrin 01 E01 7 is Expressed Selectively by Epidermal T Cells in Psoriasis and 01 E-Specific a T Cell Mediated Disruption of Anagen Hair Follicles Antibodies are Effective in Treating Murine Psoriasiform Skin Lesions K. McElwee, J. Carroll, D. Boggess, K. Silva, L. King Jr and J. Sundberg M. P. Scho¨n, J. P. Donohue, M. Scho¨n, W. W. Agace, T. Ruzicka and C. M. Parker The Jackson Laboratory, Bar Harbor, Maine; The Genetics Institute, Andover, Massachusetts; and Department of Dermatology, University of Du¨sseldorf, Germany; Division Rheumatology and Department of Dermatology, Vanderbilt University, Tennessee Immunology, Harvard Medical School, Boston, Massachusetts Alopecia areata (AA)-like hair loss can be induced in naive, normal haired recipients by transfer Mechanisms of tissue-specific T cell-localization and the role of distinct receptor molecules in the α β of skin grafts from spontaneously AA affected C3H/HeJ mice. Typically 8–10 wk after surgery, pathogenesis of psoriasis are largely unclear. The integrin 01 E01 7 is a candidate receptor to graft recipients will develop ventral and then dorsal hair loss. This simple technique has previously mediate the binding of T cells to epidermal keratinocytes, as it is expressed by lymphocytes in or been suggested as a suitable model to define the dynamics of AA after the disease activation event, adjacent to other epithelia and because it is known to bind to E-cadherin expressed on epithelial cells. in advance of overt hair loss, and during initial stages of clinical hair loss. Interestingly, within eight of eight psoriatic lesions, 53.3% (SD ϭ 9.7), but only 5.7% (SD ϭ 2.0) α β α β Skin grafts from AA-affected C3H/HeJ mice were transferred to 28 normal haired recipients. At of dermal T cells expressed 01 E01 7 (P Ͻ 0.0001). Furthermore, 01 E01 7 expression was weeks zero, 2, 4, 6, 8, 10, and 12, four grafted mice were sacrificed and skin taken for histology, observed on both CD4ϩ and CD8ϩ T cells by two-color immunofluorescent staining. α β immunohistology, electron microscopy, and RNA extraction. Gene analysis using chips for both To assess a potential functional role of 01 E01 7 in the pathogenesis of hyperproliferative 250 and 6500 genes was used to screen obvious RNA expression changes. Quantitative changes inflammatory skin lesions, we utilized a T cell-based murine model of psoriasis resulting from in specific genes were then analyzed using RNase protection analysis. Twenty-eight sham grafted transfer of minor histocompatibility antigen mismatched CD4ϩ/CD45RBhi T cells into recipient α β mice were used as controls and similarly processed. mice. Similar to human psoriasis, strong expression of 01 E01 7 was seen on T cells within the Histology showed localized inflammation of recipient anagen hair follicles immediately adjacent epidermis of these murine psoriasiform lesions. When scid/scid-recipients were treated with the α to the graft site 2–4 wk after surgery. By 6 wk anagen hair follicles proximal and distal to the 01 E-specific monoclonal antibody M290, the psoriasiform lesions were dramatically improved as graft site were inflamed. By 10 wk, inflammation was at its maximum and coincided with overt compared to an isotype-matched control antibody or untreated mice (P Ͻ 0.001). α β hair loss. Immunohistology showed inflammatory cells were predominantly CD4ϩ and CD8ϩ T Based upon these observations, 01 E01 7 integrin is expressed on intraepidermal T cells in psoriasis cells. Electron microscopy showed activated inflammatory cells closely associated with disruption and in murine psoriasiform skin lesions, where it presumably mediates binding to E-cadherin. In α of root sheaths. Gene expression analysis showed several genes to be significantly up or down addition, 01 E-specific antibodies were effective in treating a murine hyperproliferative inflammat- α regulated 8 wk after graft surgery. The genes expressed at the onset of hair loss were consistent ory skin disorder. These observations suggest that 01 E may be a therapeutic target in the treatment with a Th1 cell type inflammatory response. of hyperproliferative inflammatory skin disorders, such as psoriasis. This AA induction and propagation by skin grafting from affected C3H/HeJ mice and gene expression analysis are powerful tools to study the onset and therapy of AA.

729 730 Differential Amplification of Distinct C5a-Receptor Pathways in Normodense and Hypodense The Antigen Presenting Molecule CD1d is Expressed on Normal Human Keratinocytes (KCs) Eosinophils of Patients with Atopic Dermatitis D. Jullien, S. Porcelli, B. Nickoloff, B. Bonish and A. Claudy S. Dichmann, U. Zimpfer, W. Czech, E. Scho¨pf and J. Norgauer Department of Dermatology, Hopital E. Herriot, Lyon, France, and Divison of Rheumatology, Department of Dermatology, University of Freiburg, Freiburg, Germany Brigham and Women’s Hospital, Boston, Massachusetts, and Department of Pathology, Loyola Eosinophils play a central role in the pathogenesis of atopic dermatitis. In these patients two University, Chicago, Illinois different types of eosinophils with distinct density can be isolated. The normodense cells represent A subset of CD4ϩ or CD4–CD8– human T cells expressing an invariant V01α24J01αQ TCR- the huge majority in count, whereas the hypodense eosinophils are characterized by higher effector alpha chain, have been implicated both in the control of autoimmune disease and response to activity such as production of reactive oxygen species. To understand this altered functional tumors. These T cells which represent an average of 1/500 peripheral blood lymphocytes are responsiveness C5a-induced signal pathways and effector function in hypo-and normodense cells CD1d-restricted and therefore could play a major role in those tissues which express CD1d. have been analyzed. Intracellular Ca2ϩ-measurements revealed that C5a triggered identical Human CD1d was previously believed to be confined to intestinal epithelia. In this study, we responses in both cell types. Lipid studies by thinlayer chromatography followed by deacylation investigated whether CD1d was also expressed on skin epithelial cell-derived normal and and high pressure liquid chromatography (HPLC) showed production of phosphatidylinositol immortalized KCs (i.e. HaCaT cells) 3,4,5-triphosphate (PIP3) indicating activation of a novel intracellular signal pathway in eosinophils CD1d expression by multipassaged human KCs and HaCaT cells was assessed at both the mRNA involving phosphatidylinositol 4,5 biphosphate-3-kinase (PI4,5-P2-3-kinase). Direct comparison and protein level. Using highly specific primer sets, CD1d mRNA was constitutively detected in of PIP3 production in normo-and hypodense eosinophils demonstrated significant differences in both KCs and HaCaT cells, but not in cultured fibroblasts using RT-PCR, which was confirmed this intracellular signal cascade corresponding to the different effector activities. To further analyze in CD1d-transfected CIR B cells, but not mock-infected cells. Omission of RT from reaction the function of PI4,5-P2-3-kinase in eosinophils, the influence of the PI4,5-P2-3-kinase inhibitors mixture produced negative results. CD1d protein was also demonstrated on western blot analysis wortmannin and LY294002 on production of reactive oxygen species was studied. Both compounds in KCs and CIR-CD1d cell extracts but not in the CIR-mock cells, nor in 3T3 cells. The size inhibited with similar concentration dependency C5a-induced formation of PIP3 and production of protein expressed by KCs (approximately 48 kDa) was distinct from the previously reported of reactive oxygen metabolites. In summary these data showed for the first time the involvement size of CD1d molecules expressed on intestinal epithelial cells (37 kDa) suggesting that in KCs of PI4,5-P2-3-kinase in the production of reactive oxygen metabolites in eosinophils and differential CD1d is glycosylated. Immunostaining of normal human skin and KCs grown as monolayers on amplification of C5a-receptor signal pathways in hypodense and normodense eosinophils. Lab Tek Chamber slides using 2 different mAbs specific for CD1d revealed plasma membrane staining. Cell surface expression was confirmed by flow cytometry, and pre treatment of KCs and HaCaT cells with IFN-01γ (103 U per ml; 24 or 48 h) increased CD1d expression compared to isotype control staining. In conclusion, CD1d is not restricted to intestinal epithelium, but can be expressed by epidermal KCs. It appears to be differentially glycosylated and KC CD1d cell surface expression was inducible by IFN-01γ. Cutaneous immunohomeostasis may be regulated by a subset of peripheral blood derived CD1d-restricted T cells interacting with benign and/or malignant keratinocytes.

731 732 Effective Therapy of Murine A20 Tumors with A20-Specific T Helper 1 Cells Role of Cytolytic T Cells (CTL) in Leprosy O. Egeter, R. Mocikat, K. Ghoreschi and M. Ro¨cken M. Ochoa, P. Sieling and R. Modlin Department of Dermatology LMU, GSF-Department of for Immunology, Munich, Germany Division of Dermatology, and Department of Microbiology and Immunology, UCLA School of We studied the role of CD4ϩ-T-cells (Th) in anti tumor immunity using in vitro generated A20- Medicine, Los Angeles, California specific Th1 that produced high amounts of IFN-01γ and little IL-4 upon stimulation with A20 CTL are an important component of host immunity against intracellular pathogens. One mechanism tumor cells. We first analyzed their efficacy in tumor prevention. BALB/C mice received a lethal of cytotoxicity may have a special role in host defense, the granule exocytosis pathway. CTL with dose of A20 cells and a single injection of Th1. While 100% of the control mice died in about cytotoxic granules release the pore forming molecule perforin and granulysin, an antimicrobial 35 d, 70% of the Th1 treated mice were protected. No signs of autoimmunity were observed on protein which can kill intracellular pathogens including mycobacteria. To understand the role of autopsy after 300 d. Next we tested the A20-specific Th1 for therapy of established tumors. Mice cytolytic T lymphocytes against leprosy, we determined the distribution and frequency of proteins with a A20 tumor load of about 102 colony forming units per spleen received one injection of characteristics of CTL in skin lesions across the spectrum of the disease. Using PCR analysis, we Th1. Surprisingly a single Th1 injection cured up to 70% of the mice. Freshly isolated splenocytes found that tuberculoid patients, the resistant form of the disease, expressed granulysin and perforin from Th1 treated tumor bearing mice produced IFN-01γ when stimulated in vitro with A20 cells mRNA, whereas lepromatous patients expressed only perforin mRNA. Immunohistologic analysis showing that the transfered T cells were not tolerized by the tumor. Thus, efficient immunity revealed that the frequency of granulysin ϩ cells was greatest in the tuberculoid group. CD4ϩ T against A20 lymphoma can be established by adoptive transfer of tumor specific Th1. This suggests cells lines derived from tuberculoid leprosy lesions were to be cytolytic for antigen-pulsed targets that adoptively transfered Th1 provide a new promising strategy for therapy of established tumors. and by immunohistology expressed perforin and granulysin. The presence of perforin and granulysin in CTL in leprosy lesions is likely to represent a mechanism of antimicrobial host defense in skin. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 645

733 734 Dendritic Cell Migration to Regional Lymph Nodes Correlates to their State of Maturation Transendothelial Migration of Polymorphonuclear Leukocytes is Activated in the Patients with A. Mehling, M. Labeur, B. Pers, B. Roters, S. Beissert, T. Schwarz and S. Grabbe Generalized Pustular Psoriasis Department of Dermatology, University of Mu¨nster, Mu¨nster Germany T. Matsumura, K. Sato-Matsumura, T. Yokota, H. Kobayashi and A. Ohkawara Dendritic cells (DC) play a crucial role in initiating immune responses by migrating from sites of Department of Dermatology, Hokkaido University School of Medicine, Sapporo, Japan antigen capture to draining lymph nodes (LN) where they present antigen to naive T cells. The Massive infiltration of polymorphonuclear leukocytes (PMNL) to the epidermis is one of the main immunotherapeutic potential of DC is currently under active investigation with DC being pathological features of generalized pustular psoriasis (GPP). To elucidate the pathogenesis of administered preferentially via subcutaneous (s.c.) injection. As transport of the antigen to the LN PMNL infiltration in GPP, we evaluated transendothelial migration of PMNL from GPP patients is a key event in the generation of immunity, the capacity of DC to migrate to regional LN after using newly developed in vitro assay system. Porcine collagen gel was mixed with 5x Iscove’s s.c. injection was investigated and correlated to their state of differentiation. DC were generated modified Dulbecco’s medium at the ratio of 4:1. After adjustment of pH, 200 µl per well of the from murine bone marrow using the following combinations of cytokines: (i) GM-CSF; (ii) GM- mixed solution was polymerized in eight-well chamber slide glasses. Endothelial cells were cultured CSF ϩ IL4; (iii) GM-CSF ϩ IL4 ϩ IL10; (iv) GM-CSF ϩ IL4 ϩ TNF01α, (v) GM-CSF ϩ on the gel until confluency, and then stimulated with TNF for 4 h. After washing with PBS, IL4 ϩ LPS and (vi) GM-CSF ϩ IL4 ϩ CD40L, yielding DC of immature (i), intermediate (i– PMNL from controls or GPP patients were loaded in each well, incubated at 37°C for 2 h, and iv) and mature (v, vi) stages of differentiation. DC were labeled with the fluorescent dyes DiIC16 then fixed with 4% paraformaldehyde. Optical sections were taken at the levels of endothelial cell (5 µM), CFDA-SE (5 µM) or PKH2–2 (1 µM), and injected s.c. into footpads of mice. Two monolayer (0 µm level) and 20 µmor40µm inside the gel using a confocal laser scan microscope. days later, regional LN were extracted and analyzed by flow cytometry to monitor DC migration. The number of PMNL in each level was determined by the mean of five randomly selected fields In contrast to the other dyes, DiIC16 did not impair DC migration in vitro and exhibited minimal at each level. In GPP patients without active symptoms, transmigration of PMNL across cytotoxicity, and was therefore used for subsequent experiments. In general, 500,000 cells were nonstimulated endothelial cell monolayer was almost the same as controls, whereas transmigration injected s.c. into the hind footpad of syngeneic mice. In all experiments, DiIC16-labeled DC were across TNF-stimulated monolayers was significantly increased in GPP patients. On the other hand, clearly detectable in LN by flow cytometry. Remarkably, however, the majority (Ͼ90%) of the PMNL from active GPP patients migrated across both TNF-stimulated and nonstimulated cells remained at the site of injection, maintaining their viability. No labeled cells were found in endothelial cell monolayers significantly more than controls. Our data showed activated transendo- regional LN upon injection of dead DC, whereas several tumor cell lines migrated to LN with thelial migration of PMNL in GPP patients, which might be involved in the pathogenesis of GPP. equal or even better efficiency than DC. Mature DC generated in the presence of CD40L were superior to immature DC propagated with GM-CSF only in their capacity to migrate to regional LN. Most of the migrating cells were found in popliteal LN and almost no dyed cells were detected in inguinal LN, suggesting that DC did not migrate beyond the first LN. Cell surface expression of various adhesion molecules (CD11a, CD11b, CD18, CD49d, CD49f, CD62 L, PSGL-1) decreased during maturation, with the exception of CD11c and ICAM-1, which were upregulated upon DC maturation. However, preincubation with none of these antibodies prior to injection enhanced DC migration to LN. These experiments suggest that s.c. injected DC home rather inefficiently to regional LN and that mature DC are superior to immature DC in their migratory capacity.

735 736 Dual Function of CD44 Variant Isoform V3 in Allergic and Delayed Type Hypersensitivity The Induction of Apoptosis Via FasR/CD95 in Melanoma Cell Lines is Associated with Low Reactions Expression of bcl-2 and bcl-x and is modulated by interferon-01γ S. Seiter,*† P. Engel,* W. Tilgen† and M. Zo¨ller* S. Ugurel, S. Seiter, G. Rappl, A. Stark, W. Tilgen and U. Reinhold *Department of Dermatology, University of the Saarland, †Department of Tumor Progression Department of Dermatology, The Saarland University Hospital, Homburg/Saar, Germany and Immune Defense, German Cancer Research Center, Heidelberg, Germany Different types of tumor cells have been shown to escape immune recognition by constitutive In allergic alterations of human skin the majority of infiltrated leukocytes express CD44v3, but resistance to FasR/CD95-mediated apoptosis. We tested an unselected panel of 11 melanoma cell no other CD44 variant isoform. As CD44 isoforms have been described to influence leukocyte lines for FasR/CD95-sensitivity and the corresponding expression of FasR/CD95, FasL, bcl-2, migration and activation, it bacame of interest to evaluate whether CD44v3 facilitated leukocyte bcl-x, bcl-xS, bax and FLIP proteins. Despite detection of FasR/CD95 cell surface expression in activation or homing into the skin. We answered the question in a DTH model by comparing nine out of 11 cell lines tested, only three melanoma cell lines were sensitive to anti-FasR/CD95 the effect of anti-CD44v3 with the effect of anti-CD44s and CD44v10, both are known to mAb-induced cell death. Apoptosis-related proteins FasL, bcl-2, bcl-x, bcl-xS and bax were found suppress DTH reactions. Anti-CD44v3, too, mitigated the DTH reaction in DNFB sensitized to be heterogenously expressed in different melanoma cell lines tested. The susceptibility of and challenged mice. However, the seemingly similar effects of CD44 isoform specific antibodies melanoma cells to anti-FasR/CD95 mAb-mediated apoptosis was associated with low protein resulted from a distinct modulation of response. CD44v3 and CD44v10 are distinctly expressed expression of both, bcl-2 and bcl-x. The level of FasR/CD95 cell surtace expression in melanoma on subpopulations of activated leukocytes. CD44v3 is expressed predominantly on CD4ϩ cells cells was no indicator for FasR/CD95-sensitivity. FLIP protein was detectable in seven out of 11 and monocytes, CD44v10 is a markker of activated B cells and monocytes. Anti-CD44s mainly cell lines, but showed no correlation to FasR/CD95 sensitivity. Furthermore we could show, that influenced IL-2 and IFN01γ espression. Anti-CD44v3 and anti-CD44v10 preferentially modulated interferon (IFN)-01γ but not IFN-01γ is able to increase the susceptibility of sensitive cell lines expression of TNF01α and IL-12. By co-culturing leukocyte subpopulations it became apparent and to induce FasR/CD95-sensitivity in resistant melanoma cell lines, accompanied by an that anti-CD44v10 interfered with monocyte activation, whereas anti-CD44v3 blocked a T upregulation of the protein expression level of FasR/CD95 and/or bcl-xS. helper–monocyte interaction. Only in anti-CD44v3 treated animals the effect was more pronounced in the infiltrate than in the draining lymph nodes. The transfer of leukocytes into DNFB sensitized and lethally irradiated mice revealed that, in fact, leukocyte extravasation was strongly inhibited by anti-CD44v3. Thus, mitigation of a DTH reaction by anti-CD44v3 and anti-CD44v10 is based on distinct mechanisms which rely partly on the engagement of different leukocyte subsets and partly on an interference with leukocyte extravasation.

737 738 Withdrawn Hierarchy and Context of Epitopes Influence the Protective Immune Response against Tumor Cells A. Makki, G. Weidt, L. Lefrançois* and P. K. Srivastava Center for Immunotherapy of Cancer and Infectious Diseases, and *Division of Rheumatic Diseases, University of Connecticut Health Center, Farmington, Conneticut We have used EL4 cells and their ovalbumin (OVA) transfected counterpart E.G7 to study the hierarchy of tumor rejection antigens. Immunization with EL4 induced cytotoxic T lymphocytes (CTL) against EL4 and E.G7, in an in vitro T cell cytotoxicity assay. In contrast, vaccination with E.G7 induced lytic activity against E.G7, but not against EL4, showing that OVA epitopes are dominant over the endogenous EL4 epitopes in the E.G7 cell line. Consistent with this result, immunization with irradiated EL4 cells could confer complete protection against EL4 tumor growth and partial protection against E.G7 tumors, whereas irradiated E.G7 cells did not protect against a subsequent challenge with EL4. Surprisingly, vaccination with E.G7 did not lead to rejection of E.G7 tumors although it induced OVA-specific CTL. Growth of E.G7 tumors in mice immunized with irradiated E.G7 cells could not be explained by loss of OVA expression in vivo since five of six E.G7 tumors growing in E.G7 immunized mice were recognized by OVA- specific CTL in vitro. To exclude that E.G7 has undergone some OVA-unrelated alteration, that led to lower immunogenicity of these cells, we used an ovalbumin loss variant of E.G7. Like EL4, this variant partially protected against E.G7, indicating that the inability of irradiated E.G7 cells to protect against E.G7 tumors is a direct result of the OVA expression. In contrast to the vaccination with E.G7 cells, infection of mice with an OVA-expressing recombinant vesicular stomatitis virus (VSV-OVA) led to rejection of E.G7 tumors. We are currently questioning why a vaccination with ovalbumin in the context of tumor cells does not induce protective immunity in vivo against EL4 or E.G7, even though it elicits CTL, whereas ovalbumin in the context of a viral infection elicits a protective immune response against E.G7. 646 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

739 740 Attempted Development of a Monoclonal Antibody to a Novel Adhesion Molecule Epidermal HLA Class I Variable Chain Expression is Down-regulated in Chronic Plaque and D. A. Jones, C. W. Smith, L. J. Picker, L. Thurmon, M. Estrella and L. V. McIntire Guttate Psoriasis Institute of Biosciences and Bioengineering, Rice University, Houston, Texas; Department of A. Rowe, J. P. Schilling, E. Mallon and C. B. Bunker Pediatrics, Baylor College of Medicine, Houston, Texas; Department of Pathology, UT South- Skin Treatment and Research Trust Laboratory, Department of Dermatology (Chelsea and western Medical Center, Dallas, Texas Westminster Hospital), Imperial College School of Medicine, London, UK Adhesion of leukocytes to endothelial cells under flow conditions begins with a rolling interaction The MHC (major histocompatability) class I HLA (human leucocyte antigen) Cw*0602 allele is which is mediated primarily by selectins. In previous studies we have determined that stimulation strongly associated with chronic plaque psoriasis, HIV-associated psoriasis and guttate psoriasis. of endothelial cells for 24 h with IL-1 results in rolling adhesion of T cells and neutrophils which Genetic analysis of chronic plaque psoriasis has also demonstrated close linkage of psoriasis to the appears to be independent of the three known selectins but similar in several respects to adhesion position of the HLA C locus on chromosome 6. These data imply that HLA class I molecules, mediated by E- and P-selectin. We have further determined that stimulation with a combination and in particular HLA-Cw*0602 are involved in the pathogenesis of psoriasis. However, we know of IL-1 and IL-4 produces no adhesion under flow conditions but induces similar expression of little about the roles of these molecules in the development and maintenance of psoriatic lesions. known adhesion molecules. In this study, we used that difference as the basis for screening of We have used anti-HLA class I antibodies with fluorescent secondary antibodies to investigate monoclonal antibodies in an attempt to isolate the molecule responsible for the adhesion. Cultured HLA class I expression in lesional and nonlesional chronic plaque (CPP) (n ϭ 13) and guttate human umbilical vein endothelial cells (HUVECs) stimulated for 24 h with IL-1 were used for psoriatic (GP) skin (n ϭ 9) as compared with normal skin (n ϭ 7). In normal skin, HLA class I immunization. We used three different immunization protocols for a total of 12 polyethylene immunoreactivity (IR) was found on a number of cell types, but was strongest on the epidermal glycol fusions. Hybridomas were screened by adding supernatant to 96 well plates containing cell surface and on the vascular endothelium. As expected, a bright IR dermal infiltrate was IL-1-stimulated HUVEC monolayers followed by secondary antibody conjugated to alkaline present in both lesional CPP and GP skin, and in smaller quantities in nonlesional skin of both phosphatase. Positive clones were amplified and rescreened using HUVECs stimulated with IL-1, types of psoriasis. In both CPP and GP lesions HLA class I IR was substantially decreased both IL-1 and IL-4, or nothing. We found a total of 51 clones which bind IL-1-stimulated throughout the epidermis. There was also a reduced intensity of IR in the basal and immediately HUVECs, 31 of these also bind unstimulated HUVECs. Unfortunately, all 51 also bind to IL-1 suprabasal keratinocytes in both CPP and GP nonlesional skin. ϩ IL-4-stimulated HUVECs, and so are unlikely to recognize the adhesion molecule we hypothesize Such down-regulation of HLA class I expression in CPP and GP skin has implications for antigen is responsible for the observed difference in adhesion between these stimulation conditions presenting function and lymphocyte activity in the pathogenesis of psoriasis.

741 742 Direct Delivery of Antigenic Peptide into Lymphatic Organs as a Novel and Efficient Pulsing Neutrophil CD11b, L-selectin and FcIgA Receptor Function in Patients with Dermatitis Herpeti- Procedure of Dendritic Cells In Vivo formis (DH) A. C. Ha¨ffner, F. Koch, K. Zepter, R. Dummer, G. Burg and T. M. Ku¨ndig A. D. Smith, R. D. Streilein and R. P. Hall III Department of Dermatology, University Hospital of Zu¨rich, Zu¨rich, Switzerland Durham VA and Duke University Medical Centers, Durham, North Carolina CD8ϩ T cell responses are efficiently induced by antigenic peptides presented by dendritic cells DH patients have cutaneous Ig A deposits, an associated gluten sensitive enteropathy (GSE), and, (DCs). Current antiviral and antitumoural vaccination strategies employ DC progenitors from when untreated, develop blistering skin lesions characterized by neutrophil (PMN) infiltration at blood that have been extracorporally enriched, cultured in the presence of distinct cytokines and dermal papillary tips. Mechanisms which control PMN accumulation in the skin of DH patients pulsed with antigenic peptides before being reinjected into the patient. In contrast to these labor- are not understood fully. We have hypothesized that the mucosal immune response primes PMNs, and cost-intensive in vitro procedures the presented study conceives lymphoid organs as culture and that local factors in the skin allow them to be activated and migrate into the skin. We have dishes already containing abundant numbers of mature dendritic cells for in situ peptide labeling. shown that PMNs from DH patients on gluten containing diets (GCD) but without lesions, In fact, direct injection of peptide efficiently pulsed DCs and generated specific CTL responses express increased CD11b levels, and normal levels of L-selectin. We hypothesized that PMNs in which mediated antiviral and antitumoral protection. This in vivo pulsing method ‘‘short cuts’’ patients without lesions but on a GCD, would also show increased FcIgA receptor function; and currently used in vitro strategies and may prove useful in the immunotherapy of cancer and that when clinical lesions develop, PMNs would shed surface L-selectin, but show no change in infectious disease and is currently tested in a phase I clinical trial for immunotherapy of melanoma. CD11b expression or FcIgA receptor function. PMN FcIgA receptor function was studied in treated DH patients (n ϭ 25) and normal subjects (n ϭ 12). In addition, five DH patients on medication with no skin lesions, were studied for PMN surface expression of L-selectin, CD11b, anti-IL-8 receptors (CXCR-1, CXCR-2), and for FcIgA receptor function. Patients were then taken off therapy, allowed to develop skin lesions, and PMNs re-evaluated. EDTA-treated whole blood was obtained, placed immediately on ice, and analyzed for cell surface expression of L- selectin, CD11b, CXCR-1, and CXCR-2 using murine antihuman monoclonal antibodies. PMNs were analyzed by FACS analysis, and geometric mean channel fluorescence (GMCF) calculated. FcIgA receptor function was estimated by incubating PMNs with biotin-labeled human monoclonal IgA, and assessing binding via FACS analysis. PMN FcIgA receptors in DH patients showed a significantly increased capacity to bind IgA. (GMCF post IgA incubation/pre IgA incubation: Nls ϭ 1.44; DH ϭ 1.97; P ϭ 0.05, paired t-test). On treatment and lesion free, PMN L-selectin GMCF was significantly greater than after lesion development (GMCF: No lesions ϭ 339; lesions ϭ 226; P ϭ 0.018, paired t-test). No change in FcIgA receptor function, or in CXCR-1 and CXCR-2 expression were observed with and without lesions. PMNs from DH patients on GCD show increased CD11b and Fc IgA receptor function, suggesting they are ‘‘primed’’, probably secondary to the GSE. These results support the hypothesis that development of lesions in DH patients is the result of activation of ‘‘primed’’ PMNs locally as evidenced by decreased L- selectin expression.

743 744 Differential Expression of ICAM-1 and VCAM-1 by Psoriatic Microvascular Endothelial Cells: Gene Gun Inoculation with Plasmid Encoding Granulocyte/Macrophage Colony Stimulating Effect of Novel Retinoid Compounds Factor (GM-CSF) Increases MHC-IIϩ Dermal Dendritic Cells G. Herron, S. Michel, L. Romero, D. Zhang, I. Pelisson, I. Auzanneau, D. Lao, M. Demarchez B. Steimer, R. Tigelaar, J. Brandsma* and M. Girardi and M. Prunieras Department of Dermatology, and Comp. Med., *Yale University of School of Medicine, New Department of Dermatology, Stanford University, Stanford, California and CIRD Galderma Haven, Conneticut Laboratories, Sophia Antipolis, France Intradermal injection of recombinant GM-CSF protein into mouse skin has been shown to locally Psoriasis is characterized by epidermal cell hyperproliferation, dermal microvascular cell activation increase numbers of dermal putative antigen presenting cells (APCs). We studied the effects of and increased dermal capillaries with extensive branching. We tested whether endothelial cells gene gun inoculation with plasmid DNA encoding GM-CSF (pGM-CSF). (B10.A 01ϫ AKR)F1 from lesional skin of psoriatic patients maintained an activated phenotype in vitro before and after mice were inoculated with DNA-coated 1.6 µm gold particles using an Accell gene gun device treatment with retinoid receptor ligands. Isolation and growth of dermal microvascular cells at 250 psi pressure. Control mice received DNA-free gold particles. Both groups were compared (DMEC) from normal adult control skin and neonatal foreskin resulted in cells exhibiting typical to naı¨ve mice. Experimental mice each received two adjacent inoculations with 0.4 mg gold beads epitheliod morphology and expression of CD31; however, cells isolated under identical conditions coated with 0.2 µg pGM-CSF nonanagen razor-shaven abdominal skin. Control mice each from lesional skin of 15 different psoriatic patients exhibited a spindle-shaped morphology with received inoculations with 0.4 mg gold beads alone. After 72 h, 1 cm2 areas of inoculated skin high expression of myofibroblast markers. Addition of VEGF to culture media resulted in the growth were harvested for frozen section staining by standard immunohistochemistry using a biotin- of psoriatic microvascular endothelial cells (PMEC) exhibiting normal epitheliod morphology, high conjugated antibody directed against MHC-class II protein I-A(k). Images of sections were digitally expression of CD31, low myofibroblast markers and expression of RARa,b,g and RXRa retinoid scanned (Adobe Photoshop v5.0) and computer analyzed (NIH Image Analyzer v1.57) for numbers receptors. FACS and quantitative RT-PCR analysis of immuno-purified CD31(ϩ) PMEC (n ϭ of positively staining cells per unit area. Mice inoculated with GM-CSF plasmid DNA showed a 6) showed 20% higher expression of ICAM-1 versus control DMEC (n ϭ 3) under basal conditions, statistically significant increase in MHC-IIϩ dermal dentritic cells relative to both naive mice whereas, basal VCAM-1 expression was similar. TNF01α stimulation increased both ICAM-1 (P ϭ 0.002; Mann–Whitney U-test) and to control mice inoculated with gold beads alone (P ϭ and VCAM-1 expression in PMEC and DMEC to a similar degree; however, preincubation with 0.03). Control mice also showed a significant increase in dentritic cells relative to naı¨ve mice retinoids had a differential effect on PMEC. Whereas, all trans retinoic acid, and both a novel (P ϭ 0.03). Thus, gene gun inoculation with pGM-CSF is an effective strategy to increase dermal RARg agonist and an anti-AP-1 compound specifically decreased ICAM and VCAM expression dentritic cells, and this augmentation results from more than the effects of accelerated gold particles. in DMEC these compounds had no effect on PMEC. In addition, a RARa antagonist decreased Although, the relative contribution of plasmid DNA (i.e. containing CpG motifs) versus the VCAM transcripts in PMEC with no effect on DMEC. These results show that VEGF is a survival encoded GM-CSF protein has yet to be determined, priming with pGM-CSF may prove an factor for PMEC and that ICAM-1 and VCAM-1 expression in these cells is differentially regulated effective strategy at boosting local APCs and resulting immune responses when used prior to by retinoid compounds. vaccination with antigen-endcoded DNA plasmids. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 647

745 746 Psoriasis is Clinically and Histologically Improved by Treatment with a Humanized Anti-CD11a Natural Antibodies to Skin in Normal Individuals Monoclonal Antibody (hu1124): Results of a Multicenter, Multiple Ascending Dose Study P. Kirschner-Weinfeld, D. Jiao, Z. Xie, S. Jamal, E. Kelman and J-C. Bystryn A. Gottlieb, J. Krueger,* A. Abdulghani, H. Rashid, A. Sherr, G. Solodkina, J. Ruckle,† N. Ronald O. Perelman Department of Dermatology, NYU School of Medicine, New York, New Year Lowe,‡ S. Clark,§ R. Bright,ϩ C. McCall,** W. Shapiro,†† D. Thompson,‡‡ R. Dedrick,§§ Evidence has accumulated that a network of natural autoantibodies may play a role in immune K. Starkoϩϩ and M. Garovoy§§ self-regulation. Such antibodies have been shown to multiple tissues, but little is known about UMDNJ-RWJ Medical School, New Brunswick, New Jersey; *Rockefeller University, New their presence to skin components other than keratin. We investigated the frequency and specificity York, New York; †North-west Kinetics, Tacoma, Washington; ‡Santa Monica, California; of natural abs to skin. §Longmont, MT; ϩPRI, Palo Alto, California; **Emory, Atlanta, Georgia; ††San Diego, Forty randomly selected blood bank sera were tested by immunoblotting for IgG antibodies to California; ‡‡Milwaukee, Wisconsin; §§Xoma Corp., Berkeley, California; ϩϩGenentech, San soluble extracts of fresh normal human epidermis and cultured dermal fibroblasts and melanocytes, Francisco, California prepared by mechanical homogenization and ultracentrifugation in Tris-SDS. Antibodies to each Psoriasis is a T cell mediated disorder. CD11a (LFA-1) is a leucocyte integrin which plays a major of the three skin extracts were present in all subjects and were directed to multiple antigens (2– role in T cell activation and emigration into skin. In a previous, multicenter, single-dose study, 21 antigens for each subject) ranging in molecular weight from 31 to Ͼ200 kDa. Based on their clinical improvement, decreased numbers of epidermal T cells and keratinocyte ICAM-1 expression molecular weight, Ͼ50% of these antibodies were to antigens other than keratin. The antibody in plaque biopsies were observed after hu1124 treatment. In this report, 39 patients with moderate- responses were heterogeneous, with each person responding to a unique combination of antigens. to-severe psoriasis were treated with seven doses of hu1124 administered intravenously at 0.1 mg The frequency and magnitude of antibodies to each skin extract were similar, but the pattern of per kg every other week (Group 1); 0.1 mg per kg weekly (Group 2); 0.3 mg per kg weekly antibodies to each extract was unique. Similar results were obtained for 15 normal individuals (Group 3); 0.3, 0.4 then 0.6 mg per kg for the remaining weeks (Group 4); 0.3, 0.4, 0.6 then tested for IgM antibodies to skin. 1.0 mg per kg for the remaining weeks (Group 5). hu1124 was well tolerated and a clear dose These results indicate that natural antibodies to major components of skin are present in all response relationship was observed both clinically and histologically. In Groups 1 and 2, no individuals and that each individual has a unique repertoire of antiskin antibodies. The physiologic significant clinical or histologic response was observed; CD11a was not blocked in psoriatic function of these antibodies, and their role in immune regulation and disease processes, remains plaques. At day 42, in Groups 3, 4, and 5, the mean decrease in PASI scores were 34%, 34%, and to be determined. 52%, respectively. eight of 12 (Group 3), three of six (Group 4), four of four (Group 5) patients demonstrated histological decrease in epidermal thickness, epidermal T cell infiltration and ICAM- 1 expression (day 56). CD11a in plaques was blocked and down modulated on PBMC. PBMC, isolated from one patient in Group 4 on day 56, failed to be activated by either OKT3 or PHA in vitro. Responsiveness was detected by day 70. These data suggest that clinical and histologic improvement induced by hu1124 may be the result of decreased T cell activation, T cell emigration into skin and T cell cytokine production in vivo.

747 748 Chemical Carcinogenesis Resistant FVB/K14/IL-101α Transgenic Mice Show Enhanced Immune Expression of Distinct Cytokine Patterns in Acute and Chronic Dermatoborreliosis Responses to Epicutaneous Antigen R. Muellegger,*† G. McHugh,† R. Ruthazer,† B. Binder,* H. Kerl* and A. Steere† J. Murphy, N. Tamura and T. Kupper *Department of Dermatology, University of Graz, Graz, Austria; †Division of Rheumatology/ Harvard Skin Disease Research Center, BWH, Boston, Massachusetts Immunology, Tufts University School of Medicine, Boston, Massachusetts FVB/N transgenic mice that overexpress murine 17 kDa IL-101α (under K14 control) in the Erythema migrans (EM) is the characteristic skin manifestation of acute Lyme borreliosis (LB), basal layer of the epidermis develop a mild spontaneous inflammatory skin disease, characterized whereas acrodermatitis chronica atrophicans (ACA) is the typical cutaneous manifestation of by hair loss, scaling, and papulosquamous lesions (Groves, 1995). These K14/IL-101α Tg mice chronic LB. Cytokines are inflammatory mediators and regulators of the immune response that are completely protected against carcinoma induction in the two-stage DMBA/PMA skin have been shown to influence the course of Lyme arthritis and neuroborreliosis. The present study carcinogenesis protocol (three experiments, n ϭ 32, t Ͼ 24 mos., no carcinomas to date), even was undertaken to analyze the expression of pro-and anti-inflammatory cytokines in the skin on the highly susceptible FVB/N background (100% of nontransgenic littermates develop lethal lesions of patients with acute or chronic dermatoborreliosis. SCC). The majority of papillomas and carcinomas induced by this protocol contain a consensus Punch biopsies, fixed in formalin and embedded in paraffin, were obtained in 42 patients with H-ras codon 61 mutation; a peptide derived from this mutant H-ras sequence is an immunogenic EM and 27 with ACA. In situ hybridization with specific riboprobes was used to determine the ligand for MHC Class I in at least one MHC haplotype (H-2b). As a first step to investigating expression of mRNA for each of 5 pro-inflammatory (IL-101β, TNF-01α, IL-6, IFN-01γ, and the contribution of the immune system to this protective effect in our K14/IL-101α mice, we IL-2) and two anti-inflammatory cytokines (IL-10 and IL-4) in the skin samples. tested their capacity to mount a CHS response to the contact sensitizer oxazolone (Ox). Naive In the 42 patients with EM, there was expression primarily of mRNA for pro-inflammatory IL-101α transgenic mice exhibit significantly enhanced (Ͼ50%) ear swelling relative to the FVB cytokines in skin lesions, particularly the T cell-derived cytokine IFN-01γ [39 of 42 patients littermates, using 0.5% Ox sensitization and 1% Ox challenge. Peak ear swelling for both Tg and (93%), median percentage of mRNA signals per total of inflammatory cells (PSI) of 7.4%]. In control littermates occurred at 2 d postchallenge. The enhanced ear swelling response of the K14/ addition, in the 15 patients with extracutaneous symptoms, such as headache, myalgias, and fever, IL-101α Tg mice was prolonged, and was sustained through day 14 postsensitization. At higher the EM lesions had expression of mRNA for the pro-inflammatory, macrophage-derived cytokines, sensitizing doses of 1% and 2% Ox, the peak ear swelling response of the transgenic mice continued IL-101β, TNF-01α, and IL-6. In contrast, in the 27 patients with ACA, a more restricted pattern to increase significantly, while that of the littermate non-Tg controls did not increase further. of cytokine expression was found consisting only of mRNA for TNF-01α [24 of 27 patients When titers of both Ox specific IgM and IgG antibodies were measured, the K14/IL-101α Tg (89%), PSI of 4.8%] and the anti-inflammatory cytokine, IL-4 [19 of 27 patients (70%), PSI of 1.8%]. mice had significantly higher (more than 10-fold) levels when measured at 5 d after sensitization, EM is characterized primarily by the expression of pro-inflammatory cytokines, particularly IFN- compared to Ox sensitized, non-Tg littermates. Taken together, these results demonstrate that 01γ which may be important in control of the spirochetal infection. In contrast, ACA is constitutively elevated levels of IL-101α in epidermis enhance both humoral and cell mediated characterized by an unusual, restricted pattern of pro- and anti-inflammatory cytokine expression responses to an antigen encountered through the epidermis. The relationship of this enhanced that may be less effective in spirochetal killing. cutaneous immunity to the observed resistance to chemical carcinogenesis remains to be determined.

749 750 Critical Role of Neutrophils in the Pathogenesis of Murine Flaky Skin (fsn) Lesions Identification of Dendritic Cells as a Physiological Source of 5-Oxo-eicosatetraenoic Acid M. Scho¨n, R. Kubitza, I. Kruse, G. Wienrich, D. Denzer and M. P. Scho¨n U. Zimpfer, S. Dichmann, J. Simon, E. Scho¨pf and J. Norgauer Department of Dermatology, University of Du¨sseldorf, Bayer AG, Wuppertal, Germany Department of Dermatology, University of Freiburg, Freiburg, Germany Although neutrophil infiltration and epidermal microabscess formation is a hallmark feature of The arachidonic acid metabolite 5-oxo-eicosatetraenoic acid (5-oxo-ETE) is a potent chemotaxin psoriasis, their role in the pathogenesis of hyperproliferative inflammatory skin lesions is poorly for neutrophils and eosinophils. Until now, several unphysiologic stimuli like calcium ionophor understood. As neutrophil infiltration and microabscess formation are mirrored within the skin of A23187 and phorbol myristate acetate (PMA) are known to stimulate the production of 5-oxo- flaky skin (fsn/fsn) mice, neutrophil distribution was analyzed in CBY(fsn/fsn) mice and their ETE in different subtypes of leukocytes. the aim of this study was to identify a physiological normal littermates, and their in vivo role assessed by three complementary approaches: first, source of 5-oxo-ETE synthesis by lipid extraction techniques and high-performance liquid neutrophil function was blocked by the cytotoxic RB6-8C5 mAb (anti-Ly-6G) depleting Ͼ95% chromatography (HPLC). Although the mentioned unphysiologic stimuli provoked synthesis of of neutrophils. Second, GM-CSF, a cytokine stimulating neutrophils by rapidly upregulating 5-oxo-ETE in neutrophils and eosinophils, multiple incubation experiments with different α surface expression of integrin 01 M (CD11b), was inhibited by the neutralizing MP1-22E9 mAb. physiological stimuli such as C5a, PAF, RANTES, IL-3, IL-5, GM-CSF and 5-hydroxy- α β Third, integrin 01 M01 2, which mediates neutrophil extravasation and epidermal localization eicosatetraenoic acid (5-HETE) revealed no noticable production of 5-oxo-ETE. in contrast to through binding to ICAM-1, was inhibited by the M1/70 mAb. these data, human dendritic cells were able to synthesize this metabolite in the presence of 5- Neutrophils were almost completely cleared from the skin of fsn/fsn mice by the RB6-8C5 mAb, HETE in a time and concentration dependent manner. This condition seems to be rather specific, α β while the reduction of neutrophils was less pronounced when 01 M01 2 or GM-CSF were since the stimulation of dendritic cells with other stimuli such as C5a, PAF, RANTES, PMA and blocked. Interestingly, the epidermal thickness of fsn/fsn mice treated with anti-Ly-6G was calcium ionophor A23187 revealed no synthesis of 5-oxo-ETE. in summary these data identified dramatically reduced by 55.6% as compared to isotype-matched controls [0.67 mm (Ϯ0.16) vs dendritic cells as the only yet known physiological source of 5-oxo-ETE. This suggests a regulatory 0.29 mm (Ϯ0.08), p Ͻ 0.0001). This was accompanied by a marked reduction of epidermotrophic function of dendritic cells in the induction of inflammatory neutrophil and eosinophil infiltration T cells and by decreased expression of proinflammatory cytokines, such as TNF01α and IL-101α. caused by arachidonic acid metabolites. In contrast, the reduction of epidermal thickness was less pronounced in fsn/fsn mice treated with α anti-01 M (14.7%, p Ͻ 0.05) or anti-GM-CSF (21.5%, p Ͻ 0.01). In conclusion, neutrophils appear to ply a pivotal role in the pathogenesis of fsn/fsn lesions, α β suggesting a similar role in psoriasis. The 01 M01 2 integrin and GM-CSF, at least in part, mediate this pathogenic role. However, additional or compensatory mechanisms also appear to contribute to neutrophil localization in fsn/fsn lesions. 648 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

751 752 Aspects of the Immune Response in Pyoderma Gangrenosum Localization of Interleukin-101β mRNA and Expression of Interleukin-101β Protein in Mouse V. Feier, A. Koreck and D. Drugarin Skin Following Topical Exposure to the Chemical Allergen, Oxazolone Department of Dermatology, Department of Immunology, University of Medicine Timisoara, F. Kermani, M. S. Flint and S. A. M. Hotchkiss Romania Dermal Toxicology Research Group, Molecular Toxicology Section, Sir Alexander Fleming Pyoderma gangrenosum is an uncommon, ulcerative skin disease. The etiology and pathogenesis Building, Imperial College School of Medicine, London, UK; and NIOSH, Morgantown, are still not understood but abnormalities of the immune system may have a great role. West Virginia The purpose of our study was to evaluate some aspects of the immune response in patients with Allergens capable of inducing contact sensitivity cause changes in levels of epidermal cytokine pyoderma gangrenosum. The study was performed on 10 patients with pyoderma gangrenosum mRNAs. in mice, one of the earliest cytokines to be upregulated following sensitization is hospitalized in the Dermatology Clinic Timisoara between 1995 and 1998. The diagnosis was interleukin-101β 01([capiota]L-101β), the major epidermal cellular source of which is the made on clinical and histological criteria and our investigations found no evidence of an associate Langerhans cell. the aim of the present study was to investigate the induction and localization of disease (inflammatory bowel disease, arthritis, hematological disorder or malign disease). The IL-101β protein and mRNA expression, in mouse skin, following exposure to the chemical methods used for the immunological investigation were immunophenotyping of peripheral blood allergen, oxazolone. the kinetics and localization of murine IL-101β were studied at the mRNA lymphocytes with a Becton-Dickinson flowcytometer and determination of serum levels of the level using in situ hybridization (ISH) and at the protein level by enzyme-linked immunosorbant following cytokines: IL-1, IL-2, IL-4, IL-6, IFN01γ (ELISA technique). At the time of these assay (ELISA). Female BALB/c mice (n ϭ 4) were topically exposed to 1% oxazolone dissolved investigations no patient received immunosuppressive therapy. in acetone:olive oil vehicle, with control mice being exposed to vehicle alone. Flank skin, excised The results indicate normal values for lymphocyte populations and subpopulations. The expression at various times following oxazolone or vehicle exposure, was divided and half the sample was of activation marker CD25 (low affinity receptor for IL-2) on T cells was elevated in patients analyzed for IL-101β mRNA by ISH and half was analyzed for IL-101β protein by ELISA. IL- with pyoderma gangrenosum (12.53% Ϯ 2.45%) comparative with healthy controls (2.08% Ϯ 101β mRNA was expressed, constitutively, in naı¨ve (untreated) and vehicle-treated skin, with 1.06%) and the expression of HLA-DR molecules was in normal range. We found slowly elevated mRNA localized in some hair follicles and sebaceous glands; no mRNA was detected in the levels of proinflamatory cytokines IL-1 (3.3 Ϯ 1.75 pg per mg) and IL-6 (5.3 Ϯ 2.08 pg per ml). epidermis. Following exposure to oxazolone for 5–15 min, upregulation of IL-101β mRNA was the serum levels of IL-2 (62 Ϯ 8.72 pg per mg) and IFN01γ (21.6 Ϯ 4.51 pg per mg) were high observed in the dermis, hair follicles and sebaceous glands with evidence of epidermal expression. and the level of IL-4 was 4.5 Ϯ 2.6 pg per mg. Beyond 15 min oxazolone exposure, the pattern of IL-101β mRNA expression was indistinguishable The elevated levels of IL-2 and IFN01γ together with the increased expression of CD25 on T from control. Analysis of whole skin homgenates by ELISA, demonstrated IL-101β protein to be lymphocytes support the active implication of T cells, especially Th2 subset in the evolution of present, constitutively, in mice exposed to vehicle alone (33.1 Ϯ 1.9 pg per ml, mean Ϯ SEM, pyoderma gangrenosum. This can be considered a base for the administration of immunosuppressive n ϭ 4) and in naı¨ve mice (36.3 Ϯ 0.92 pg per ml). In oxazolone-treated animals, IL-101β protein drugs with predominant effect on T lymphocytes for the therapy of pyoderma gangrenosum. increased at 30 min following treatment (77.0 Ϯ 2.9 pg per ml), decreased at 1 h (48.1 Ϯ 17.8 pg per ml), fell below the limit of detection (20 pg per ml) at 2 h, before returning to near constitutive levels at 4 h (32.6 Ϯ 9.0 pg per ml) and 24 h (45.3 Ϯ 10.8 pg per ml). There was no change in IL-101β protein levels in vehicle-treated or naı¨ve animals over this time period. in conclusion, IL-101β mRNA and protein expression were upregulated in mouse skin following sensitization with the contact allergen, oxazolone. Early upregulation of IL-101β suggests this cytokine may play a key role in the initial stages of skin sensitization.

753 754 Effects of Transforming Growth Factor beta-Inhibition on Disease Progression in a Murine Constitutive Expression of CX3C Chemokine Fractalkine in Mouse Skin Sclerodermatous Graft Versus Host Disease Model for Scleroderma N. Kanazawa,*† K. Yoneda,* K. Tashiro,‡ K. Inaba,§ Y. Miyachi* and T. Honjo† L. McCormick, E. Tootell, Y. Zhang and A. Gilliam *Department of Dermatology, †Department of Medical Chemistry, Faculty of Medicine, ‡Center Department of Dermatology, Case Western Reserve University and University Hospitals of for Molecular Biology and Genetics, §Department of Zoology, Faculty of Science, Kyoto Cleveland, Cleveland, Ohio University, Kyoto, Japan Scleroderma is a chronic debilitating autoimmune fibrosing disorder of unknown etiology with Fractalkine is a unique membrane anchored CX3C chemokine. We isolated this chemokine from immune dysregulation and overproduction of dermal type I collagen. Growth factors, including mouse bone marrow-derived mature dendritic cells (DCs) and showed that in the lymph node it TGF01β1, produced by activated monocytes infiltrating skin are thought to initiate and perpetuate is constitutively expressed in T cell area DCs and can attract T cells. So fractalkine may play a abnormal collagen synthesis and cause skin thickening. Until now, there has been no animal model role in recruitment of T cells to T cell area DCs in the resting state. But considering that which closely mimics human scleroderma. We are currently utilizing a murine model in which fractalkine mRNA is expressed in rather ubiquitous tissues containing brain, heart, lung, kidney lethally irradiated BALB/C (H-2d) mice transplanted with bone marrow and spleen cells from and short intestine and that in the brain this chemokine is reported to be synthesized by neurons B10.D2 (H-2d) mice develop sclerodermatous graft vs. host disease (Scl GVHD), which recapitulates and attract microglias, fractalkine may possibly be expressed in other nonhematopoietic cells. So early scleroderma. We have established a time course of the skin and lung changes in Scl GVHD we examined which cells express fractalkine in the skin. Using RT-PCR method mRNA of mice compared with control mice receiving syngeneic bone marrow. Scl GVHD mice exhibit fractalkine and its receptor CX3CR1 was detected in the skin. the result of in situ hybridization the following characteristics after bone marrow transplantation (BMT): skin thickening by day 20, revealed that the specific signal for fractalkine antisense RNA was diffusely detected in the spinous upregulated production of TGF01β1-mRNA by day 6, upregulated production of collagen layer of footpad epidermis, not in the basal and granular layer. We found that mouse keratinocyte mRNA by day 20, lung fibrosis by day 20, and an increase in the number of CD11b-expressing cell line PAM212 cells, which is known to secret chemotactic factor for dendritic epidermal T mononuclear cells by day 14, which is most pronounced on day 20. Further, our data indicate cells, also synthesized this chemokine. the cells expressing CX3CR1 and recruited in the resting that the inhibition of TGF01β1 through the administration of antipan TGF01β antibodies at days skin are now under investigation. 1 and 6 post-BMT can block macrophage infiltration into skin and prevent lung fibrosis and skin thickening. These data confirm the central role of TGF01β in the development of Scl GVHD and identify TGF01β as an effective target for disease treatment or prevention which may eventually extend to the treatment of human scleroderma.

755 756 Differential Regulation of Prostaglandin E2 Release in Atopic Dermatitis Patients by the Inducible IgM Antiendothelial Cell Antibodies from Patients with Behcet’s Disease Can Induce the Cyclooxygenase Isoform Expression of Adhesion Molecules and the Adhesion of T Lymphocytes to Endothelial Cells D. J. Gordon and C. A. Holden K. Lee, H. Chung, S. Lee and D. Bang Dermatology Department, St. Helier Hospital, Surrey, UK Department of Dermatology, Yonsei University College of Medicine, Ajou University School of Abnormal release of Prostaglandin E2 (PGE2) from monocytes may be a fundamental defect in Medicine, Seoul, Korea atopic dermatitis (AD). We have examined the role of the constitutive (COX-1) and inducible The pathogenesis of Behcet’s disease (BD) has not been clarified. Circulating antibodies to (COX-2) isoforms of cyclooxygenase on PGE2 release from peripheral blood mononuclear cells endothelial cells(AECA) have been demonstrated in a variety of autoimmune diseases with vascular (PBMC) in short-term cell culture. Stimulated and unstimulated PBMC from AD patients (n ϭ pathology, including BD. In this study, we have demonstrated the effects of the AECA in sera of 7) and nonatopic controls (n ϭ 9) were cultured alone and with COX-1 or COX-2 specific BD patients on the expression of endothelial cell adhesion molecule and the adhesion of T inhibitors at three concentrations for 2 h. Cell supernatants were analysed for PGE2 by ELISA. lymphocytes to vascular endothelial cells. IgM autoantibodies to human dermal microvascular Intracellular fluorescence staining of the two COX isoforms was performed in PBMC from 14 endothelial cells (HDMEC) were detected in 42 of 75 sera from Behcet’s disease patients. subjects (seven AD patients and seven controls) to estimate the presence or absence of COX-2 in Pretreatment of HDMEC with AECA-positive BD sera or IgM purified from these sera led to fresh PBMC. Stimulation of PBMC produced an increase in PGE2 release from both groups, an increase or an induction in the expression of intercellular cell adhesion molecule-1 (ICAM-1), compared to unstimulated cells. the increase was clearly significant in AD (P ϭ 0.00014), but was vascular cell adhesion molecule-1 (VCAM-1) and E-selectin on cultured HDMEC. After small and nonsignificant in controls, suggesting increased COX-2 activity in AD PBMC. the stimulation of HDMEC with AECA, the expression of ICAM-1 and VCAM-1 on HDMEC selective inhibition of COX-2 compared to COX-1 was greater in AD at the two highest inhibitor increased significantly at 4 h, reaching a peak at 16 h. Expression of E-selectin was induced at 1 concentrations (P ϭ 0.01), but not at any concentration in the controls. Flow cytometry indicated h after stimulation with a peak at 4 h and decreased thereafter. Adherence of T lymphocytes to the absence of COX-2 in freshly prepared PBMC from both groups. the rate of COX-2 expression HDMEC increased significantly after stimulation with AECA in sera of BD patients. Transfer of and activity in stimulated AD PBMC was increased. This may be of importance in the regulation HDMEC-conditioned media after pretreatment with AECA and immunodepletion of IgM of PGE2 release in AD. demonstrated the presence of transferable activity that mimicked the effects of AECA. Treatment with neutralizing anticytokine antibodies indicated that IL-101α and TNF01α, generated by the HDMEC in response to AECA, was involved in the upregulation of endothelial cell adhesion molecules and T lymphocytes adhesion. These results show that IgM AECA can play a pathogenic role in BD by activating endothelial cells, in part due to autocrine or paracrine actions of IL- 101α or TNF01α. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 649

757 758 Co-Expression of the Eotaxin Receptor CCR3 and CD 30 in Cutaneous T Cell Lymphoma Isolation from Nail Plates of a Unique Antimicrobial Peptide(s) Active Against Candida Albicans M. Kleinhans, M. Gilliet, G. Burg and F. Nestle R. A. Dorschner and R. L. Gallo Department of Dermatology, University Hospital Zurich, Switzerland Department of Dermatology, Boston Children’s Hospital and Harvard Medical School, Boston, Cutaneous T cell lymphomas are a group of skin seeking lymphoproliferative disorders. Few is Massachusetts known about mechanisms involved in skin specific homing of malignant T cells. Recently Nails are exposed to harsh environmental conditions and multiple potential pathogens, yet typically chemokine/chemokine receptor interactions have been implicated in homing of leukocytes to resist infection. This microbial resistance occurs despite the inaccessibility of the nail plate to sites of tissue inflammation. The CC chemokine receptor CCR3 is selectively expressed on TH- classical immune mechanisms. We hypothesized that antimicrobial peptides may contribute to the 2 cells and therefore a marker for a polarization of the cytokine secretion pattern of a given T immune protection of nails. This system of antimicrobial defense is the primary immune system cell. CD 30 is an additional surface marker implicated in the differentiation of T cells towards a for insects and plants, but has only recently been described in mammalian skin. to investigate TH-2 type of cytokine secretion. the CC chemokines eotaxin, RANTES and the monocyte whether antimicrobial peptides exist in nails, porcine nail plates were extracted and proteins chemotactic proteins MCP-3 and MCP-4 are all ligands for the CC chemokine receptor CCR3, purified based on their ability to inhibit the growth of Candida albicans. Potent anticandida activity but only the chemokine eotaxin is selective for CCR3. We investigated tissue samples of 21 was detected in aqueous solutions from acidic extracts prepared with 1 M HCl, 1% trifluoroacetic patients with CTCL for in situ expression of the chemokine receptor CCR3 and its ligands by acid. Anti-candida activity bound cationic exchange columns and eluted at buffer salt concentrations immunohistochemistry. Additionally we investigated freshly isolated tumor cells for CCR 3 and greater than 1 M NaCl. Activity was also bound by C18 hydrophobic reversed phase columns, CD 30 expression by flowcytometry. Seven out of eight CD30 positive CTCL expressed CCR and eluted with acetonitrile concentrations greater than 30%. This anticandida molecule was 3. in contrast, no CCR 3 expression was found in skin tissue samples from CD 30 negative temperature resistant (100°C for 30 min) and protease sensitive (Papain or Protinase K). the crude CTCL (n ϭ 13). Additionally, coexpression of CCR 3 and CD 30 could be demonstrated by nail extract inhibited growth at protein concentrations as low as 20 µg per ml, but did not inhibit flow cytometry in freshly isolated tumor cell suspensions of CD 30 positive CTCL. Furthermore gram negative bacterial proliferation. Therefore, a hydrophobic, cationic protein is present in nails we were able to detect the CC chemokine eotaxin in lesional skin of CD 30 positive CTCL. that is able to protect against growth of C. albicans. The characteristics of this protein suggest it is Our data suggest an important role for CCR3 and its ligand eotaxin in the recruitment of a novel antimicrobial peptide and its activity provides further evidence for an innate defense malignant T cells in CD30 positive CTCL and establish a link between migration of lymphoma mechanism that protects against infection without inflammation. cells into the skin and their functional state as TH-2 cells.

759 760 The Correlation Between the Expression of Cytokines and Transglutaminase 1 and 2 in Human Circulating Thl and Th2 Type Cytokines in Psoriasis Head and Neck Squamous Cell Carcinoma Cell Lines (HNSCCs) S. Rose, A. Abdulghani, K. Raja, R. Chandramouli and A. Gottlieb C. Lee, S. Kim,* P. Steinert* and M. Park Division of Clinical Pharmacology, Clinical Research Center, University of Medicine and Dentistry Oral and Pharyngeal Cancer Branch, National Institute of Dental Research; *Laboratory of Skin of New Jersey-Robert Wood Johnson Medical School, New Brunswick, New Jersey Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes Over expression of several proinflammatory Th 1-type cytokines has been demonstrated in various of Health, Bethesda, Maryland immune disorders including psoriasis and is believed to be of pathogenic significance. However, Transglutaminases (TGases) have been proposed to be involved in both differentiation and cellular recent studies show that monocyte and other T cell derived cytokines (Th2-type) also play an remodeling during malignant cell invasion and metastasis in skin diseases. to understand the important role in the Thl, Th2 balance. to investigate whether a predominant typel or type2 is physiological regulatory mechanisms of TGases, we screened for the effector cytokines for TGases present in psoriasis patients, we determined the profile of Thl and Th2 cytokines in the sera of in HNSCCs as a model system. We analyzed expression levels of TGases and various cytokines 55 patients (57.69% with Ͼ1 0% body surface area involvement) with psoriasis and 15 patients in three sets of HNSCCs from primary and metastatic regions. Here we report that TGase 1 with other (13 noninflammatory and two inflammatory) dermatoses. We employed a highly expression was down-regulated in all HNSCCs compared with normal keratinocytes, but TGase sensitive ELISA assay to measure the levels of circulating serum IL2, IL4, IL10, IL12, IFN01γ and 2 expression varied from cell line to cell line. However, those variations are not associated with TNF01α. A significant increase of the typel cytokines IL2 (96.96 vs 15.86 pg per ml, P Ͻ 0.01) apoptosis. We found that the elevated expression of TGase 2 correlated with increased TNF-01α and IFNg (333.44 vs 116.20 pg per ml, P Ͻ 0.02) was observed in the psoriasis group compared expression suggesting important role(s) of TGase 2 under TNF-01α control in the keratinocytes. to controls. However, the type 2 interleukin, IL10, was significantly decreased (16.30 vs 40.86 pg Interestingly the increased expression of TGase 1 correlated with elevated expression of TGF-01β per ml, P Ͻ 0.002) in the psoriasis group. the circulating monocyte-derived cytokines TNF01α and inversely correlated with PDGF expression in HNSCCs. Most other factors including IL-10, and IL12 were also decreased significantly (17.63 vs 24.53 pg per ml, P Ͻ 0.03 and 7.73 vs 25.20 M-CSF, IL-101α, IL-101β, and IL-6 had no direct relationship with expression of TGases 1 and pg per ml, P Ͻ 0.00003). Although, production of Thl -type cytokines was increased in psoriasis, 2. These data suggest that TGase 1 may be related to cellular differentiation under TGF-01β we found no corresponding decrease in circulating IL4, a Th2-type cytokine. No correlation was control and TGase 2 may be related to antiproliferation under TNF-01α signaling. Further study found between age, sex, duration of psoriasis and serum levels of cytokines. However, a marginal will reveal the cytokine responding element(s) potentially involved in the regulation of the increase of IL2 was related although not significantly (r ϭ 2.438) to disease severity in patients expression of TGases. with psoriasis (Ͼ10% of the body surface area involved). the change in the Thl, Th2 balance (as a result of selective induction of IL2 and IFN01γ) may mirror psoriatic activity. Our data support the important proinflammatory role of the Thl type cytokines in the clinical manifestations of psoriasis. In diseases such as psoriasis, where a specific causative agent/antigen has not yet been identified, acting on the Thl/Th2 balance might represent an alternative approach to control disease activity, e.g. with IL10.

761 762 Mechanisms for Control of Antimicrobial Gene and Peptide Expression in Cutaneous Defense Cytokine Pathways in Contact Hypersensitivity: Role of IL-10 and IL-18 V. Pestonjamasp, K. Huttner and R. Gallo B. Wang, L. Zhuang, H. Fujisawa, C. Feliciani,* G. Shivji, H. Suzuki, P. Toto,* C. Dinarello† Department of Dermatology, Children’s Hospital, Harvard Medical School, Boston, Massachusetts and D. Sauder Cathelicidins are antimicrobial peptides that are components of innate immunity. Expression of Division of Dermatology, Sunnybrook Health Science Centre, University of Toronto, Toronto, these peptides in skin enables direct bacterial killing without recruitment of inflammatory cells. Ontario, Canada; *Chieti University, Cheiti, Italy; †University of Colorado Health Sciences To understand how these peptides are controlled, the murine homologue, CRAMP was studied. Centre, Denver, Colorado Genomic structure of CRAMP consists of four exons and three introns spanning a total of about Contact hypersensitivity (CHS) is a type 1 T-cell mediated immune response to epicutaneous 4 kB. Complete sequencing of both strands of CRAMP shows a striking similarity with LL-37, haptens. Epidermal Langerhans cells, Langerhans cell-derived cytokines and keratinocyte-derived the human cathelicidin induced in contact dermatitis. Like LL-37, CRAMP is inducible. CRAMP cytokines play a significant role in the initiation of CHS. Previous studies have demonstrated that mRNA was barely detectable in unstimulated Balb MK keratinocytes, whereas it was induced IL-10, a type 2 T-cell cytokine, downregulates CHS, and that IL-10 gene knockout (KO) mice upon differentiation initiated by the addition of 2 mM Caϩϩ. the 5Ј untranslated region (5Ј mount an exaggerated CHS response. In order to investigate the mechanisms underlying this UTR) of CRAMP contains potential binding sites for multiple transcription factors, including enhanced response, we analyzed the cytokine pattern in the draining lymph nodes (DLN) during NF-kB, AP-1 and HFH2. Truncations of the CRAMP 5Ј UTR were carried out and cloned the induction phase of CHS. Mice were sensitized with DNFB, and then DLN collected at into a luciferase reporter vector. Transfection experiments using transformed keratinocytes have various time points. Cytokine mRNAs were examined by RT-PCR analysis. IL-10 KO mice suggested sequences in the 5Ј UTR which influence the CRAMP promoter activity. to study demonstrated an enhanced type 1 T-cell polarization in DLN following hapten sensitization. The regulation of CRAMP protein expression, mouse skin 24 h after injury was evaluated by mRNA for Langerhans cell-derived cytokine IL-12 p40 was significantly higher in IL-10 KO immunohistochemistry with affinity purified polyclonal antiserum. CRAMP protein was abundantly mice compared to wild-type (WT) mice. Since IL-18, a recently discovered cytokine, shares many expressed at the wound surface and to a lesser extent in superficial keratinocytes adjacent to the functional properties with IL-12, and is a dendritic cell (DC)/Langerhans cell-derived cytokine, wound. CRAMP protein was also detected in normal mouse bone marrow where it was found we analyzed its gene expression. While IL-18 mRNA was constitutively expressed in DLN, it within granulocytes. Thus CRAMP is inducible under the conditions of epithelial barrier was significantly upregulated after hapten sensitization in IL-10 KO mice and, to a less extent, in compromise and can be regulated as a component of the immune system, where it contributes to WT mice. to further investigate whether IL-18 is functionally relevant to the enhanced CHS the first line of skin defense. response, we performed IL-18 antibody blocking studies. Mice received anti-IL-18 antibody 2 h before and 24 h after hapten sensitization. Neutralization of endogenous IL-18 partly reversed the enhanced CHS in IL-10 mutant mice. Our data suggest that IL-18 is involved in the increased CHS response in IL-10 KO mice and that IL-18 plays an immunoregulatory role in the induction of CHS. 650 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

763 764 Altered Production of Multiple Cytokines in Xeroderma Pigmentosum Fibroblasts Following Production of Interleukin-18 and its Receptor in Human Keratinocytes UVB Irradiation: Implications for Cutaneous Phenotype in Xeroderma Pigmentosum J. Mee and R. Groves F. M. O’Reilly, H. Yang, J. Chang, J. C. Ansel and C. A. Armstrong Centre for Dermatology, Department of Medicine, University College London, UK Department of Dermatology, Emory University, Atlanta, Georgia Interleukin-18 (IL-18) is a recently described cytokine which, although structurally similar to IL- Xeroderma Pigmentosum (XP), an autosomal recessive disease involving defective nucleotide 1, shares many functions with IL-12. in common with IL-101β, IL-18 requires proteolytic cleavage excision repair, is characterized clinically by photosensitivity, abnormal pigmentation and multiple by caspase-1 to gain biological activity and binds IL-1Rrp1, a member of the IL-1 receptor family. skin cancers on sun-exposed skin. We postulate that abnormal UVB induction of cytokines from Although IL-18 production has been reported recently in murine keratinocytes, there is currently target cells in the skin may result in some of the phenotypic abnormalities seen in XP. We have no data on the production of this potentially important immunomodulatory molecule in human initiated these studies using primary XP fibroblasts which have been exposed to UVB. Approxi- skin and we therefore sought to determine whether IL-18 and its putative receptor were expressed mately 10% of incident UVB is capable of reaching human dermal fibroblasts. The induction of by human keratinocytes. several candidate cytokines was determined in three separate XP fibroblast strains as compared to Expression of IL-18 and IL-1Rrp1 mRNA was determined by semiquantitative RT-PCR analysis normal human dermal fibroblasts (HDF) following UVB irradiation. Cells were cultured in serum of primary human keratinocytes derived from neonatal foreskins and cultured in the presence or free medium 48 h prior to exposure to 10, 30, 50, 100 mJ UVB (FS 20 Westinghouse bulbs). absence of a variety of potential stimuli, including IL-101β and the mitogen, PMA, for 6 h. Both Culture supernatants were collected at 24 and 48 h after irradiation and analyzed by ELISA for IL-18 and IL-1Rrp1 were expressed constitutively at the mRNA level, although no significant the pro-inflammatory cytokines IL-1, IL-6 and TNF-01α; the angiogenic factors IL-8 and bFGF; increase was observed with any of the stimuli tested in keratinocytes derived from four different and the immunosuppressive cytokine IL-10. Constitutive production of IL-8 was higher in all XP donors. To determine whether IL-18 message was translated into protein, keratinocyte lysates fibroblasts when compared to HDF. Maximum induction of IL-8 and IL-6 occurred at a lower were assessed by western blot analysis. High level IL-18 immunoreactivity could be detected in dose of UVB (10 mJ) when compared to HDF. Constitutive production of TNF-01α was greater lysates derived from unstimulated keratinocytes with a molecular weight of approximately 28 kDa, in XP fibroblasts as compared to HDF, but no TNF-01α response to UVB was observed in any consistent with the precursor form of the molecule. of the cell types. Augmented UVB induction of bFGF, known also to be mitogenic for melanocytes, These results demonstrate that IL-18 is synthesized constitutively within human keratinocytes, occurred in XP fibroblasts when compared to HDF. in contrast, no IL-1 or IL-10 was observed although in unstimulated keratinocytes the protein remains in its unprocessed form intracellularly. constitutively or following UVB irradiation in XP fibroblasts or HDF. the data indicate that XP This observation parallels the keratinocyte IL-1 system, in which IL-101β is also produced but fibroblasts exhibit altered production of multiple cytokines both constitutively and following UVB not processed, and raises the question of the role of caspase-1 in keratinocytes. In view of the irradiation as compared to HDF. This suggests the possibility that UVB mediated release of potent immunoregulatory functions associated with IL-18, the presence of both ligand and receptor cytokines and growth factors from dermal fibroblasts may be responsible for some of the phenotypic on human keratinocytes may have important implications for the control of cell mediated cutaneous abnormalities in XP. inflammatory responses within the skin.

765 766 Chemokine/Chemokine Receptor Expression in Keloid and Normal Fibroblasts Mechanism of IL-12 Responsiveness in Leprosy: Cutaneous Expression and Regulation of IL-12 R. Devalaraja, C. Nirodi, L. Nanney, S. Arrindell, S. Russell, J. Trupin, T. Daniel and A. Richmond Receptor Beta Subunits Department of Veterans Affairs, Nashville, Tennessee; Departments of Cell Biology, Plastic Surgery, J. Kim, K. Uyemura, T. Rea and R. Modlin Vanderbilt University School of Medicine and Department of Microbiology, Meharry Medical Division of Dermatology and Department of Microbiology and Immunology, University of College, Nashville, Tennessee California Los Angeles School of Medicine, Los Angeles, California; Section of Dermatology, The disregulation of chemokine and chemokine receptor expression has been observed in a University of Southern California School of Medicine, Los Angeles, California number of tumor models. Keloids represent benign tumor lesions with proliferation of fibroblasts Regulation of the IL-12 signaling pathway is of critical importance for the development of Th1 and myofibroblasts with some infiltration of leukocytes. We undertook to determine whether this responses. IL-12 exerts its biological effect by binding to the IL-12R01β1/01β2 heterodimer on benign proliferation of fibroblasts which is accompanied by an upregulation of collagen synthesis activated T and NK cells, signaling through 01β2 subunit, and inducing tyrosine phosphorylation in coordination with alterations in the AP-1 family of transcription factors could involve of Stat 4. We investigated IL-12 responsiveness in leprosy, focusing on expression of IL-12R. By disregulation of CXC chemokines or chemokine receptors. Immunohistochemical staining for RT-PCR, we found that the inducible 01β2 subunit was highly expressed in 10 of 10 tuberculoid MGSA/GRO and its receptor, CXCR2, was performed on keloids, hypertrophic scars and normal lesions but only weakly expressed in 10 of 10 lepromatous lesions. In contrast, the constitutively skin samples. Staining for MGSA/GROa within the nodular, lesional areas was an infrequent but expressed IL-12R01β1 subunit was equally expressed in both tuberculoid and lepromatous lesions. occasional finding that seemed to show a positive correlation with the degree in inflammatory Next we examined the upregulation of IL-12R on PBMC from leprosy patients. Activation of infiltrates. by contrast, CXCR2 immunoreactivity was quite definitive in the majority of keloids, T-cells with Mycobacterium leprae resulted in upregulation of IL-12R01β2 expression in tuberculoid but not the hypertrophic scars or normal skin. Preliminary staining with smooth muscle actin as patients but not in lepromatous patients. in contrast, the IL-12R01β1 expression was similar in well as morphological evidence suggest that the bulk of the CXCR2 immunostaining is attributable both groups. Furthermore, gel shift analysis demonstrated that M. leprae induced Stat phosphorylation to myofibroblasts. By northern analysis of cultured keloid and normal skin fibroblasts, we were in lymphocytes from tuberculoid patients but not from lepromatous patients. Our data suggest unable to detect expression of MGSA/GRO or CXCR2 unless cultures were induced with IL- that IL-12R01β2 expression in leprosy lesions and on PBMC correlate with CMI and the presence 1. The wounding response of monolayer cultures of keloid or normal fibroblasts were examined of Th1 responses. Thus, the regulation of IL-12R01β2 expression may determine the balance of in vitro. Preliminary data indicate significant differences in the rate of wound closure between Th1 versus Th2 responses during cutaneous response to microbial pathogens. normal and keloid cells. Immunofluorescence for CXCR2 during the process of wound healing reveals an increased focal expression of the receptor at the edge of the wound. We postulate that the MGSA/GRO ligand interaction with CXCR2 facilitates this wound closure differentially in keloid and normal fibroblasts.

767 768 Human Herpesvirus 6 in Pityriasis Rosea Antibodies to Human Papillomavirus Type 5 (HPV5) in Autoimmune Bullous Diseases M. L. Placa, M. Vignoli, C. Varotti and M. Re S. Majewski, M. Favre, A. Pura, M. Olszewska, G. Orth and S. Jablonska Department of Clinical and Experimental Medicine, Division of Dermatology and Division of Department of Dermatology and Venereology, Warsaw School of Medicine, Warsaw, Poland; Microbiology, University of Bologna, Bologna, Italy Unite Mixte Inst. Pasteur/INSERM U190, Inst. Pasteur, Paris Human herpesvirus 6 (HHV-6) and human herpesvirus 7 (HHV-7) are recently identified In a recent study with the use of specific ELISA with epidermodysplasia verruciformis (EV) HPV5 herpesviruses, which cause largely diffused infections starting early in childhood. HHV-6 is the virus-like particles we found antibodies against the conformational epitopes of this EV HPV in agent of Exanthem subitum in children, while the association of HHV-7 with human diseases is about 25% of patients with psoriasis, and in over 90% potentially oncogenic HPV5 and related still not established. EV HPVs DNA were detected in the skin. In controls (atopic dermatitis, renal allograft recipients, In a recent study, the presence of HHV-7 DNA has been detected by PCR in plasma, peripheral patients with genital warts and healthy individuals) anti HPV5 antibodies were found in only 2%– blood mononuclear cells (PBMC) and lesional skin in adult patients with Pityriasis rosea (PR), an 5%. We suggested that HPV5 could play some role in autoimmune mechanisms of psoriasis, acute papulo-squamous disorder of unknown etiology which has been associated with the presence possibly as an autoantigen. Since psoriasis could be regarded as a wound healing, and HPVs of various bacterial and viral infections, thus suggesting that PR could be a clinical presentation proliferate in concert with keratinocyte proliferation, we asked whether anti-EV HPV5 antibodies of HHV-7 reactivation. appear also in the process of epidermal repair. We have chosen autoimmune bullous diseases as a In our study, we enrolled seven healthy subjects and six patients affected by acute PR. Peripheral model of reepithelialization, and we correlated the presence of anti-HPV5 antibodies with the venous blood samples were collected from all patients. Specimens were obtained from skin lesions disease-specific antibodies and the course. to find out the role of autoimmunity in generation of of all PR patients. anti-HPV5 antibodies, we studied sera from 15 patients with extensive burns. Twenty of 81 Nested PCR for the detection of HHV-7 and HHV-6 were performed from PBMC of all subjects (23.4%) sera from autoimmune bullous diseases were positive for HPV5 antibodies, i.e. in similar and from skin lesions of PR patients. frequency as in psoriasis. the percentages of positive sera were: for pemphigus foliaceus (PF) 30.4% HHV-7 DNA sequences were not detected either from PBMC samples or cutaneous specimens (seven of 23), pemphigus vulgaris (PV) 18.4% (seven of 38), bullous pemphigoid (BP) 20.4% (six in healthy individuals and PR patients. However, in one PR patient, HHV-6 DNA sequences of 25). Only one case of burn was positive for the antibody. The differences between PV and PF were detected both in PBMC and skin. might be due to more superficial epidermal changes with constant desquamation and more Our data strongly contrast with the exclusive causative role of HHV-7 in the pathogenesis of PR. extensive skin involvement in PF. Repeated studies revealed seroconversion of HPV5 antibodies On the other hand, the detection of HHV-6 in one patient, rather indicates that PR may be the in three patients, disappearance of HPV5 antibodies in one, and their persistence in seven cases, clinical outcome of infections caused by different agents. with no correlation to the activity of the disease and titers of disease-specific antibodies. Thus anti-EV HPV5 antibodies appear not to play a significant role in the pathogenesis of autoimmune bullous diseases. However, EV HPVs might be involved in the process of reepithelization, and autoimmunity could enhance formation of antiviral antibodies. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 651

769 770 ICAM-1 and MHC Class 1 Molecule Upregulation on HDMEC after Contact with HSV-Infected In Contrast to Low Virulence Candida Species, which Induce Immunoregulatory IL-12 and IL- PBMC: An Explanation for the Inflammatory Response in the Dermis of HSV-Induced 10, High Virulence Candida Induce IL-10 without IL-12 by Human Blood Monocytes Erythema Multiforme J. Xiong, M. Ghannoum, Y. Yoshida, K. Cooper and K. Kang N. Sepp, R. Hattmansdorfer, A. Glasser, C. Fu¨rhapter, M. P. Dierich, P. Fritsch and C. Larcher Center for Medical Mycology, Department of Dermatology, Case Western Reserve University, Department of Dermatology and Institute of Hygiene, University of Innsbruck, Innsbruck and University Hospitals of Cleveland, Cleveland, Ohio Erythema multiforme (v. Hebra) is currently conceived as a cell mediated immune reaction aimed Fungal infections caused by Candida (C) species, mainly C. albicans, are increasing especially in at the destruction of keratinocytes expressing HSV antigens. HSV-DNA reaches distant cutaneous immunocompromised individuals. Candida pathogenesis involves interaction with vascular and locations via blood stream, mainly by peripheral blood mononuclear cells (PBMC). However, the perivascular monocytes in tissues, and can directly induce monocyte cytokine production, as pathomechanism of HSV transfer from blood to tissue is not known. shown previously by our group. Monocytes have the potential to produce the immunoregulatory Direct infection of human microvascular endothelial cells (HDMEC) with herpes vim 1 (incubation cytokines IL-10 and IL-12; differential regulation by Candida may be a critical factor in determining 1 h with a m.o.i of 10–100, infection rate ~80%) and examination of adhesion molecules (HLA the nature of inflammatory and immunoresponse to virulence and avirulence Candidal strains. class 1, CD31, CD54, CD36, CD44) of HDMEC Ϯ addition of cytokines (IFN-01α, Ϫ01γ, Peripheral blood mononuclear cells (PBMC) were obtained from healthy volunteers’ blood by TNF-01α) resulted (4–5 h) in an increase of CD31 and CD54 surface expression and a decrease gradient centrifugation. Purified monocytes were prepared by adherence of PBMC to plastic of HLA class I molecules. Fourteen hours after infection CD31 was downregulated (even in the dishes and negative selection using a magnetic column to remove T cells, B cells, and NK cells presence of cytokines), whereas CD36, CD44 and CD54 remained unchanged. Supernatants from (purity Ͼ 90%). Monocytes were incubated with a live high virulence C. albicans (CA) and a low infected HDMEC showed no change of the cytokine pattern. virulence C. krusei (CK), at the ratio of 0.5 of Candida:monocyte, in 5% CO2 incubator at 37°C PBMC (75% infected monocytes, 30%–40% T- or B-cells) were stimulated for 24 h with PHA for 20 h. Monocytes were cultured alone as control. Cytokines in the supernatant were measured (1 µg/µl), inoculated with HSV-1 (m.o.i. 10–100) for 1 h followed by cocultivation with HDMEC by ELISA. the results shown as mean Ϯ SE are as follows: (i) Both live CA and CK significantly for 1 h. This cocultivation with HSV-infected PBMC for 1 h was able to increase HLA class I induced IL-10 production by monocytes relative to unstimulated monocytes (1001 Ϯ 354 for and CD54 surface expression on HDMEC (up to 5-fold) although only approximately 1% of EC CA, 1727 Ϯ 324 for CK vs 11 Ϯ 8 pg per ml from control monocytes, P Ͻ 0.05 n ϭ 6). (ii) were infected. UVC irradiation of infected PBMC (inactivation of HSV!) did not influence Live CA did not induce any IL-12 production by monocytes. (iii) Live CK induced significant upregulation of CD54 of EC. Northern blot analysis demonstrated the decreased HLA class I IL-12 production by monocytes vs unstimulated monocytes (31 Ϯ 12 vs 0 Ϯ 0 pg per ml, P Ͻ expression after direct infection of HDMEC, but upregulation after contact with HSV infected 0.01, n ϭ 16). In summary: high and low virulence Candida species can induce IL-10 production, PBMC. Adhesion assays with PBMC (infected and noninfected) demonstrated that this upregulation however, IL-12 production can only be induced by low virulence Candida species. These data of adhesion molecules was also functionally relevant. Our studies demonstrate that within a short indicate that one consequence of high virulence Candida species interacting with blood or tissue time HSV-infected PBMC were able to induce adhesion molecule expression relevant for an monocytes/macrophages is to activate monocytes/macrophages into a state of IL-10 production inflammatory response despite the low rate of HSV-infected HDMEC. This may explain the without monocytes/macrophages IL-12 production, which may attenuate effective host immune observed inflammatory response in the dermis wound vessels in HSV-mediated multiforme. response.

771 772 Expression of VEGFR-3 and Podoplanin Suggests a Lymphatic Endothelial Cell Origin of Kaposi’s Human Retrovirus-5 is Not Involved in the Pathogenesis of Psoriasis Sarcoma Tumor Cells E. Mallon, D. Griffiths, P. Venables, C. Bunker and R. Weiss W. Weninger, T. A. Partanen,* S. Breiteneder-Geleff,† C. Mayer, H. Kowalski,† M. Mildner, J. Department of Dermatology, Imperial College School of Medicine, Chelsea and Westminster Pammer,† M. Stu¨rzl,‡ D. Kerjaschki,† K. Alitalo* and E. Tschachler Hospital, London; Chester Beatty Laboratories, Institute of Cancer Research, London01; Kennedy DIAID, Department of Dermatology; *Institute of Clinical Pathology; University of Vienna; Institute of Rheumatology, London †Department of Pathology, University of Helsinki, Finland; ‡GSF-Forschungszentrum, Obersch- The pathogenesis of psoriasis is unknown but current opinion favours the disease being leiβheim, Germany immunologically mediated and possibly autoimmune in nature. Retroviral infection may play a Despite intensive research the exact lineage derivation of Kaposis` sarcoma (KS) tumor cells has role in the pathogenesis of autoimmune disease and molecular mimicry has been suggested as a escaped characterization so far. in the present report we investigated the expression of two markers possible mechanism for the induction of autoimmunity. The precipitating activation signal in for lymphatic endothelial cells (EC), i.e. vascular endothelial growth factor receptor-3 (VEGFR- psoriasis is unknown but candidates include infectious agents. Particles resembling retroviruses 3) and podoplanin, in AIDS and classic KS. Both antigens were strongly expressed by cells lining have previously been reported in psoriatic plaques. a novel partially characterised human exogenous irregular vascular spaces in early KS lesions and by tumor cells in advanced KS. by confocal laser retrovirus, designated human retrovirus-5 (HRV-5), has recently been described and has been microscopy we show that VEGFR-3 positive and podoplanin positive cell populations are identical implicated in the pathogenesis of inflammatory arthropathies including psoriatic arthropathy. and also express CD31. However, these cells were found to be negative for CD45, CD68, and We have investigated the role of HRV-5 in the pathogenesis of psoriasis. Skin biopsies were PAL-E, excluding their hemopoietic and blood vessel endothelial cell nature. Podoplanin protein obtained from both involved and uninvolved skin of 20 patients with chronic plaque psoriasis, 10 expression in KS tumors was confirmed by Western blot analysis. Both splice forms of VEGFR- patients with guttate psoriasis and two patients with HIV-associated psoriasis. Normal skin was 3 were found in KS tumor derived RNA by RT-PCR and northern blot analysis. In contrast to obtained from surgical specimens of 15 patients without inflammatory skin disease. In addition KS tumor cells in situ, no expression of VEGFR-3 and podoplanin was detected in any of four blood was obtained from all subjects. DNA was extracted from skin and blood using established KS-derived spindle cell cultures and in KS Y-1 cells in vitro. In conclusion, the expression of two methods; the quality of the extracted DNA was tested by polymerase chain reaction (PCR) for lymphatic EC markers on KS tumor cells strongly suggests that they are related to or even derived the single copy gene endogenous retrovirus-3 (ERV-3). HRV-5 proviral DNA was tested for by from the lymphatic EC lineage. Lack of these antigens on cells cultured from KS lesions indicates a sensitive nested PCR assay. that these cells might not represent KS tumor cells, but rather other cell types present in KS lesions. All the psoriatic and control samples tested were negative for HRV-5 proviral DNA. The results indicate that neither psoriatic skin nor blood are sites of HRV-5 viral replication and imply that HRV-5 is not involved in the pathogenesis of psoriatic skin disease.

773 774 Identification of a Novel Human Papillomavirus L1 Region in Association with Epidermodyspla- Expression of Cellular Prion-Related-Protein by Human and Bovine Keratinocytes In Situ and sia Verruciformis In Vitro D. Armstrong, A. Hood and A. Roman J. Pammer, W. Weninger* and E. Tschachler* Department of Dermatology, Department of Microbiology and Immunology, and Department of Institute of Clinical Pathology; *DIAID, Department of Dermatology; University of Vienna, Austria Pathology, Indiana University School of Medicine and the Walther Cancer Institute, Indiana- The exact routes of transmission of prion diseases frequently remain obscure. Recent reports on polis, Indiana the peripheral inoculation of prions prompted us to study the expression of PrPc in normal and Epidermodysplasia Verruciformis (EV) is a genodermatosis characterized by widespread and diseased skin and mucous membranes by immunohistochemistry and in cultured keratinocytes persistent infection with human papillomavirus (HPV). A subset of HPV types, defined in part by (KC) by western blot analysis. Whereas in normal human skin only little expression of PrPc the DNA sequence of the viral L1 gene, is associated with this rare dermatosis. We obtained protein, mainly confined to epidermal KC, was detected, this antigen was strongly expressed on biopsy samples from three members of an Icelandic family who had verrucous papules and plaques both KC and infiltrating mononuclear cells in inflammatory skin diseases. Bovine keratinocytes in consistent with EV. The clinical diagnosis was supported by the characteristic HPV cytopathic situ weakly expressed PrPc mainly confined to basal keratinocytes. This expression was distinctly effect present in the histologic sections. To determine the HPV type present in the biopsies, each upregulated in acanthotic epidermis. In contrast to human skin, the upregulation of PrPc in bovine specimen was analyzed by the polymerase chain reaction (PCR). There were no amplification skin adjacent to inflammations was only weak. In tissue culture, exposure of human KC to TGF- products obtained using degenerate consensus primers to EV-associated types. However, approxi- alpha or IFN-gamma lead to an increase of PrPc protein expression, whereas bovine PrPc could mately 100 bp, 250 bp, and 400 bp PCR products were obtained using a degenerate general HPV not be upregulated by several human cytokines. In conclusion, our data suggest that both human consensus primer pair. The amplification products were subcloned into pUC19 for DNA sequence and bovine squamous epithelial PrPc may represent an easily accessible target for prions and might analysis. This analysis revealed that one of the biopsy specimens contained a novel HPV type. therefore be the first target in peripheral infection. 652 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

775 776 Regulation of HMG-CoA Synthase Transcription by Sterols, EGF and Insulin Trancutaneous Molecular Delivery In Vivo with a Single Mechanical Pulse I. R. Harris, W. Siefken, H. Ho¨ppner and K. -P. Wittern S. Lee, N. Kollias, D. McAuliffe, T. Flotte and A. Doukas Department of Molecular Biology, PGU-Skin Research Center, Beiersdorf AG, Germany Wellman Laboratories of Photomedicine, Massachusetts General Hospital; Department of Dermato- HMG-CoA Synthase (HMG-SYN) is a key enzyme in the synthesis of cholesterol, which is logy, Harvard Medical School, Boston, Massachusetts essential for maintenance of a normal epidermis and homeostasis of the epidermal permeability The aim of the study was to investigate transcutaneous molecular delivery mediated by mechanical barrier. the promoter of HMG-SYN, as with many of the enzymes required for cholesterol pulses. A single mechanical pulse was sufficient to increase the permeability of the stratum corneum synthesis, has both sterol responsive elements (SRE) and AP-1 responsive elements. We have in animals and in humans in vivo. Molecular delivery was measured using a number of probe determined whether transcription of HMG-SYN can be regulated in human keratinocytes by molecules, such as 5-aminolevulinic acid, epinephrine, insulin, and labeled-dextran up to 70 kDa transfecting them with a luciferase reporter construct containing the promoter of HMG-SYN. molecular weight. The molecular probes were present during the mechanical pulse or applied Treatment with exogenous sterols decreased luciferase reporter activity, whereas inhibiting immediately after. In both cases molecular delivery occurred. These experiments suggest that endogenous sterol synthesis using fluvastatin increased activity. TPA, a putative AP-1 activator, transcutaneous delivery proceeds in two steps. (i) Permeabilization of the stratum corneum occurs increased activity by up to 20-fold after 2 h. Northern blotting demonstrated a change in steady- during the duration of the mechanical pulse. (ii) Molecules proximal to the stratum corneum state mRNA levels with sterols, fluvastatin and TPA ϩ cyclohexamide. An increase in luciferase diffuse passively into the epidermis under the concentration gradient. the presence of surfactants activity was also observed with 0.1 mg per ml EGF and 5 µg per ml insulin of 5- and 6-fold, during the application of the mechanical pulse delayed the recovery of the barrier function of the respectively. In summary, we have been able to demonstrate that transcription of HMG-CoA stratum corneum. Confocal and electron microscopy showed that the epidermis and dermis was synthase can be regulated by sterol levels and cytokines in human keratinocytes. the increase in normal after the application of the mechanical pulse. Furthermore, the barrier function of the HMG-SYN mRNA levels during barrier repair may be a result of cytokine stimulation or removal stratum corneum always recovered. of sterol suppression.

777 778 The Use of Noninvasive Bioengineering Techniques to Assess Skin Barrier Function Following Presence of a Physiological Keratin Oxidation Gradient in Human Forehead Stratum Corneum Application of a Chemical Probe J. Thiele,* S. Hsieh, K. Briviba and H. Sies K. Costa, A. Kligman and T. Stoudemayer *Department of Dermatology, Friedrich-Schiller University, Jena, Germany; and Institute for SKIN, Inc., Conshohocken, PA and Department of Dermatology, University of Pennsylvania, Physiological Chemistry I, Heinrich-Heine-University, Du¨sseldorf, Germany Philadelphia, Pennsylvania As the outermost skin layer and penetration barrier, the stratum corneum is constantly exposed Chemical probes are topically applied agents which are capable of eliciting specific physical to a pro-oxidative environment, including UV-irradiation, air-pollutants, and chemicals. Previously, (whealing, erythema) and neurosensory (burning, itching) reactions. The evolution and devolution we have demonstrated that stratum corneum lipids are targets of oxidative stress induced by ozone of these reactions can be used to provide information on skin barrier function. These reactions and by UVA and UVB exposure. Here, we employed a new immunoblotting technique to detect customarily have been subjectively graded. For this study, we used noninvasive bioengineering protein carbonyl groups as markers of protein oxidation in human stratum corneum obtained by techniques to quantify the response to dimethyl sulfoxide (DMSO), a well-defined enhancer of tape stripping. After lysis, protein carbonyl groups were measured by derivatization with penetration. These techniques included: diffuse reflectance spectroscopy (DRS) to assess changes dinitrophenylhydrazine, separation by SDS-PAGE, and immunoblotting using antibodies against in oxyhemoglobin, Laser Doppler perfusion imaging (LDPI) to measure changes in blood flow, dinitrophenyl groups. Human keratin 10, identified by use of specific antibodies and confirmed colorimetry for redness (expressed as the a* component of the L*a*b* color system), electrical by protein microsequencing, was demonstrated in vitro to be oxidizable by UVA irradiation, conductance (Novameter) to evaluate hydration of the horny layer, evaporimetry to measure hypochlorite, and benzoyl peroxide. Most remarkably, a keratin 10 oxidation gradient with low transepidermal water loss (TEWL) and Silflo replica analysis to visualize alterations of surface levels in the lower stratum corneum layers, and about 3-fold higher contents of carbonyl groups topography and textural features. Following a 15-min application of 5 µl DMSO, there were towards the outer layers was demonstrated in untreated forehead stratum corneum of healthy significant increases (P Ͻ 0.05) in oxyhemoglobin, blood flow, redness, hydration and diffusional volunteers (n ϭ 6). Since protein oxidation is well known to be associated with an increased water loss. Image analysis of Silflo replicas demonstrated moderate effacement of skin glyphics and susceptibility to proteases, the demonstration of a protein oxidation gradient in the human stratum lines. These changes returned to baseline 24 h later. Hence, noninvasive bioengineering techniques corneum may contribute to better understand the regulation of desquamation. allow for objective, quantitative assessment of barrier function as compared to subjective, qualitative (visual) assessments.

779 780 In Vivo Infrared Analysis of the Recovery of Sebaceous Lipids after Delipidation Rapid Antigen Delivery with Photomechanical Waves for Inducing Allergic Skin Reaction L. Brancaleon, M. Bamberg and N. Kollias S. Lee, S. Gonzalez, D. McAuliffe, N. Kollias and A. Doukas Wellman Laboratories of Photomedicine, Massachusetts General Hospital, Boston, Massachusetts Wellman Laboratories of Photomedicine, Massachusetts General Hospital, Department of Dermato- With this work we present the results of the investigation of in vivo recovery of sebum after logy, Harvard Medical School, Boston, Massachusetts delipidation of the stratum corneum. The study has been conducted on 12 healthy subjects by The goal of the study was to investigate the use of photomechanical waves for rapid delivery of employing an infrared instrument (FTIR) which combine Attenuated Total Reflection (ATR) antigens through the stratum corneum barrier and into the skin. Photomechanical waves have technology with the use of an optical fiber extension. This combination allows non invasive been shown to increase the permeability of the stratum corneum for a few minutes before the in vivo and in situ (the ATR crystal is only 3 mm in diameter) infrared investigation of the skin barrier function recovers. the advantage of photomechanical delivery is that it may be able to surface. Our detection system allows to separate the contribution of sebaceous and stratum distinguish allergic from irritant skin reaction because the delivery of antigen can be well monitored. corneum lipids to the overall infrared signal through the transfer of a layer of sebum onto the the standard technique in the differential diagnosis of contact dermatitis (CD) is patch testing probe upon contact with the skin. Compared to SC lipids, sebaceous lipids show the absorption using Finn chambers. This requires long occlusion times of ~48 h which can cause irritancy. the due to triglycerides and fatty acids. in areas with a lower concentration of sebaceous glands the morphologic features of both forms of CD are very similar on gross and microscopic examination presence of triglycerides and fatty acid is minimal. We delipidized the skin in two sites (01µ4.5 and it is difficult to differentiate one from the other. However, if the antigen can be delivered cm2) of the forehead and followed the subsequent recovery of sebum for 2 h. The two sites were, into the skin without prolonged skin exposure (i.e. £1 h), irritancy can be reduced or eliminated. respectively, delipidized with isopropylalchool (70%) and tape stripping (four strips applied for 1 The allergic skin reaction using photomechanical delivery for 1 h was compared with 1 h and 21 min each). The recovery of sebum was recorded by monitoring the infrared bands of sebaceous h occlusion in a sensitized hairless albino guinea pig model. The pigs were sensitized by intradermal lipids deposited on the probe. We also recorded the intensity of the infrared water bands in the injection of (0.01%) dinitrochlorobenzene (DNCB) and topical administration (0.1%, one week skin as a function of time. This gave us information about the process of hydration of the SC as later) of the hapten. One month later, testing for the allergic response was performed by the sebaceous lipids recovered. the recovery is equivalent to 3.8 01ϫ 10–5 mols per s. We administration of 50 µl of 0.25% DNCB. Our results show that there were allergic reactions for determined the rate of recovery and we found that triglicerides and fatty acids recover at a slightly 21 h occlusion and 1 h photomechanical delivery of the antigen. In contrast, no response was different rate (3.8 and 2.7 01ϫ 10–5 mols per s, respectively). Water tends to decrease as a function observed for 1 h occlusion with the antigen. in conclusion, the rapid delivery of antigens with of the lipid recovery in agreement with the role of the hydrophobic barrier. Our method may photomechanical waves may help in the differential diagnosis of CD which is the most common prove most useful in the detection of the effect that different treatments have on the secretion of form of occupational dermatoses. sebum and also on the effect that a number of diseases have on the functionality of sebaceous glands. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 653

781 782 Delivery of Histamine by Iontophoresis Into the Human Skin: Dosage Through Microdialysis In Vitro Cytotoxicity of ERC-9 Against Melanoma Cells Technique and Simultaneous Blood Flow Measurement T. Nakatsui, T. Salopek, J. Scott and A. McEwan C. Geveaux, T. Leroy, G. Shaker, C. Chinnery,* M. Church* and D. Van Neste Division of Dermatology, University of Alberta, Cross Cancer Institute, Edmonton, AB, Canada Skinterface spri, Tournai, Belgium; *Immunopharmacology Group, Southampton General Hos- ERC-9R (N[3-(4-morpholino)-propyl]-N-methyl-2-hydroxy-3-methyl-5-iodobenzyl-amine] pital, UK shows significant uptake and retention in melanoma tissue, and has proven useful in the in vivo The aim of this study was to determine, with the microdialysis technique, the dermal concentrations imaging of melanoma when labelled with I-123 [J Nucl Med 1998; 38(5 suppl):10]. Although of histamine delivered by iontophoresis of histamine solutions and to compare the results with ERC-9 appears to be useful as an imaging agent, its inherent cytotoxicity and potential use as a real time measurement of the skin microvascular response. therapeutic agent has not been explored. To assess the in vitro cytotoxicity of ERC-9, various cell For each subject (six females and four males), four microdialysis fibres were introduced in the lines ranging from melanoma cells (pigmented [SK-23, SK-28, MeWo] versus nonpigmented [SK- anaesthetised dermis of the flexural forearms (two sites on each forearm). Two hours later, we 24, UT]), neural crest lines (C6 & SK-NMC), an epithelial carcinoma (HeLa), and a fibroblast performed the iontophoresis (1.4 mA per cm2 for 30 s) of histamine 900 mM, 9 mM and 0.09 line were assayed using a modified MTT-microculture tetrazolium cell proliferation assay. The mM on three different sites plus a control site using bidistilled water. the fibers were perfused effects of ERC-9 on cell proliferation were compared with two agents previously shown to be with a saline solution (rate of 5 µl per min) and the dialysate was collected continuously 2 min cytotoxic to melanoma cells, dihydroxyphenylalanine (L-dopa) and N-propionyl-4-S-cysteaminyl- before and during 10 min (five samples of 2 min) after switching on the iontophoretic device. phenol (NPrCAP). ERC-9 had cytotoxic effects on all melanoma cell lines with IC50 values Simultaneously Laser-Doppler Rowmetry readings (perfusion units, PU) were recorded at the ranging from 0.095 to 0.160 mM. ERC-9 appeared to be more toxic to melanoma cells than agonist administration sites (located on the microdialysis fiber) and at 1 cm from them. NPrCAP (IC50 ϭ 0.12–1.00 mM), but was less effective in inhibiting melanoma growth than L- Despite wide interindividual variations, RIA determination of histamine concentrations in the dopa (IC50 0.016–0.075 mM); the latter, however, was nonspecific in its toxicity to cells. ERC- dialysates correlated with the applied concentrations (P Ͻ 0.05). the mean concentration of 9 had in general toxic effects on other cell lines tested, however, normal fibroblasts were relatively histamine was 19.44 nM for the control site and 62.23, 143.34 and 432.51 nM, respectively, after insensitive to the agent (IC50 ϭ 0.39 mM). The selective retention of ERC-9 by melanoma iontophoresis of histamine 0.09 mM, 9 mM and 90OmM. In the flare, there were also higher cells, its inherent cytotoxicity which can be enhanced by labelling with I-131 suggests that it may LDF values (P Ͻ 0.005) after iontophoresis of histamine 900 mM (mean Ϯ SD: 83.31 Ϯ 34.82 useful as a radiopharmaceutical in the treatment of melanoma. PU) compared to histamine 9 mM and 0.09 mM (respectively 30.91 Ϯ 40.82 and 53.61 Ϯ 57.01 PU) and control (4.42 Ϯ 14.64 PU). At all histamine administration sites, the increase of the blood flow perfusion peaked between the 1st and 2nd min, followed by a decline associated with the development of the weal. We conclude that there is a correlation between the applied concentration, the dermal concentration of histamine (as confirmed by microdialysis) and the real time Laser Doppler evaluation of the skin bioresponse at agonist administration site.

783 784 Finasteride in the Treatment of Men with Androgenetic Alopecia Using a Phototrichogram Safety Profile of Topical Keratinocyte Growth Factor-2 in Man Technique P. A. Jimenez, D. J. Odenheimer, R. C. Browar, V. Roschke and A. C. Louie D. Van Neste, V. Fuh, P. Sanchez-Pedreno, E. Lopez-Bran, H. Wolff, D. Whiting, J. Roberts, J. Human Genome Sciences, Inc., Rockville, Maryland; S. Wason, Phoenix International Life J. Stene, A. Tosti, S. Calvieri, D. Kopera, M. Guarrera, E. Prens, P. Kanojia, W. He and K. Sciences Inc., Cincinnati, Ohio; and J. G. Krueger, the Rockefeller University, New York, D. Kaufman New York Skin Study Center–Skinterface, Tournai, Belgium; Hospital Virgen de la Arrixaca, Murcia, Spain; Keratinocyte growth factor-2 (KGF-2), also described as FGF-10, is a member of the fibroblast Hospital Clinico San Carlos, Madrid, Spain; Ludwig-Maximilians-University of Munich, Munchen, growth factor family. KGF-2 is 57% identical to KGF-1/FGF-7 and specifically stimulates growth Germany; Dallas Associated Dermatologists, Dallas, Texas; North-west Cutaneous Research of epithelial cells in vitro. Animal studies have demonstrated that KGF-2 stimulates skin wound Specialists, Portland, Oregon; HAIR Technology–Skinterface, Brussels, Belgium; Universita` di healing in excisional and incisional wounds in mice, rats and rabbits. In addition, KGF-2 unlike Bologna, Bologna, Italy; Universita` degli Studi di Roma, Rome, Italy; University of Graz, Graz, other growth factors, appears to stimulate granulation tissue growth as well as epidermal closure. Austria; Universita` di Genova, Genova, Italy; Ziekenhuis Walcheren, Vlissingen, Netherlands; and The purpose of this double-blind study was to assess the safety of topical administration of multiple Merck Research Laboratories, Rahway, New Jersey doses of human recombinant KGF-2 in normal subjects. Thirty-six normal subjects were The growth of scalp hair is a cyclical process of successive phases of growth (anagen) and rest sequentially enrolled into 1–4 cohorts (0.1, 1, 10, or 100 µg dose per wound). Each cohort (telogen). In previous clinical trials, finasteride 1 mg was shown to increase scalp hair counts in a consisted of nine subjects, seven receiving topical KGF-2 and two receiving placebo every third defined area (i.e. to increase hair density) in men with androgenetic alopecia. The current study day for 19 d. On day 1, each subject received one pair of 6-mm punch-biopsy wounds on the used a phototrichogram methodology to assess the effect of finasteride 1 mg on the phases of the left and right upper buttocks. Two wounds were randomized to treatment with KGF-2 and two hair growth cycle. Two hundred and twelve men (ages 18–40 years) with androgenetic alopecia wounds were randomized to treatment with placebo. KGF-2 was well tolerated without local or at the vertex were randomized to finasteride 1 mg or placebo for 48 wk. At baseline, and at 24 systemic adverse events, or change in any laboratory parameter that was judged to be related to 2 and 48 wk of treatment, macrophotographs measuring total or anagen hair count in a 1-cm target KGF-2. Time to complete healing was similar (Ϯ19 d) for the KGF-2 and placebo-treated area at the leading edge of the vertex balding scalp were taken 3 d apart. Telogen hair count was wounds. No subject developed anti-KGF-2 antibodies. Histologic examinations from day 0 defined as the difference between the total and the anagen hair counts. At baseline, mean total biopsies showed normal skin without significant abnormalities. Day 78 re-biopsies showed normal and anagen hair counts in the target area in the finasteride group (n ϭ 93) were 200 and 124 features of wound healing without cellular atypia. Epidermal histology and thickness were similar hairs, respectively (% anagen ϭ 62%), and the anagen to telogen ratio was 1.74. In the placebo between day 0 and day 78 specimens. Ki67 immunostaining indicated higher proliferation of group (n ϭ 91), the respective values were 196 and 119 hairs (% anagen ϭ 60%) and 1.57. At keratinocytes at day 78 compared to baseline, irrespective of whether wounds were treated with week 48, patients on finasteride 1 mg had a net improvement in total and anagen hair counts of topical KGF-2 or placebo. the outcomes for tissue healing in both groups were entirely normal. 17.3 (95% C.I. 12.5–22.2) and 27.0 (95% C.I. 21.3–32.7) hairs, respectively, compared with While KGF-2 may have increased cell hyperplasia transiently in wound healing, there was no placebo (P Ͻ 0.001). Furthermore, compared with placebo, the anagen to telogen ratio improved by 47% (95% C.I. 27%–68%) with finasteride (P Ͻ 0.001). Finasteride 1 mg per d for 48 wk evident residual effect at day 78 postwounding. All tissue changes observed were compatible with increased both total and anagen hair count. The improvement in the anagen to telogen ratio is normal wound healing. direct evidence that finasteride 1 mg per d promotes conversion of hair follicles into the anagen These results showed that topical KGF-2 was well tolerated and support the evaluation of KGF- phase. This beneficial effect on the hair cycle with finasteride 1 mg implies favorable consequences 2 in patients with pathologic wounds. on cosmetically important aspects of hair quality (thickness, length, growth rate, growth duration and/or pigmentation), contributing to the visible improvements in hair growth observed in treated patients.

785 786 Differential Expression of Substance P in Perifollicular Scalp Blood Vessels and Nerves after Complete Healing of Venous Leg Ulcer Patients Treated with Compression Therapy can be Topical Therapy with Capsaicin 0.075% (Zostrix HP) in Controls and Patients with Extensive Predicted by Average Healing Rates at 4 wk Alopecia Areata (AA) M. L. Sabolinski and M. V. Falanga* M. Ericson, K. Binstock, A. Guanche and M. Hordinsky Organogenesis, Inc., Canton, Massachusetts and *Boston University School of Medicine, Roger Department of Dermatology, University of Minnesota, Mpls. Minnesota Williams Medical Center, Providence, Rhode Island The purpose of this study was to examine perifollicular nerve function after topical therapy with In a prospective trial, 136 venous leg ulcer patients were treated with compression therapy weekly Zostrix HP. Exposure of primary afferent sensory neurons to capsaicin, the active agent in Zostrix for up to 24 wk. Wound tracings were obtained at each visit, and computerized planimetry was HP, causes neurogenic inflammation and the release of SP. used to evaluate ulcer area. Healing rates (HR) in cm per wk were calculated using an established Two adult females with extensive AA, a disease characterized by collapsed perifollicular nerves, formula (01A/p, A ϭ Area, p ϭ perimeter). at study completion, the average HRs for all (n ϭ applied Zostrix HP to the entire scalp and two control patients, applied drug to a shaved selected 136), healed (n ϭ 65), and nonhealed (n ϭ 71) patients were determined at 4, 12, and 24 wk. site on the scalp, q 12 h for 21 d. Four-nun scalp punch biopsies were taken pretreatment and at For all patients the average HRs were 0.0859, 0.0730, and 0.0691, respectively. Wounds that Days 1 and 21. Samples were triple labeled with (i) mouse antipan-neuronal protein gene product completely healed during the study had increased average HRs of 0.1206 at 4 wk, 0.1069 at 12 9.5 (PGP 9.5) and Cy 3.18, (ii) rabbit anti-SP and Cy 5.18, and (iii) FITC-labeled Ulex wk, and 0.1017 at 24 wk. Wounds that did not heal, showed decreased average HRs of 0.0542 europaeus 1 agglutinin (UEA 1). An additional set of control biopsies was stained with rabbit at 4 wk, 0.0418 at 12 wk, and 0.0392 at 24 wk. Within each group of patients, all patients, anticalcitoningene-related-peptide (CGRP), PGP 9.5, and UEA 1. In-focus images of optical healers, and nonhealers, results of HRs were consistent throughout the period of observation. sections were captured by laser scanning confocal microscopy. Computer reconstruction of The average HRs of healers compared to nonhealers were statistically significant at 4 (P ϭ 0.005), captured images yielded three-dimensional views of the architecture and interaction of scalp 12 (P ϭ 0.0006), and 24 (P ϭ 0.0007) wk. These data show that the average HRs at 4 wk are nerves, blood vessels, and SP or CGRP expression. able to predict clinical outcomes. Rapid identification of patients who are unlikely to respond to We found perifollicular nerves and blood vessels of controls responded differently to a 21-d course standard compression Alone may allow for earlier interventions with alternative therapies. of Zostrix HP compared to those of AA patients. PGP 9.5 expression in epidermal nerves all but disappeared in the AA patients in contrast to controls. At Day 21, perifollicular nerves surrounding the bulge/bulb region of miniaturized AA hair follicles were outlined with the immunoreactants to SP whereas in the controls, the prominant finding was the expression of SP within the peribulbar vasculature. in controls, CGRP staining was unchanged after the 21 d course of Zostrix HP. Interestingly, both AA patients experienced vellus scalp hair regrowth during therapy. The enhanced presence of SP in AA perifollicular nerves and the induction of vellus hair regrowth could be related. The selective enhancement of SP within normal peribulbar blood vessels may reflect a unique property of this population of endothelial cells. 654 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

787 788 Correlation Between Cyclic Adenosine Monophosphate Phosphodiesterase Inhibition and Clinical Topical Triamcinolone and Tacrolimus Differ in Their In Vivo Effects on Langerhan’s Cells During Anti-Irritancy the Treatment of Atopic Dermatitis E. Goyarts, T. Mammone, N. Muizzuddin, K. Marenus, D. Maes K. Gordon, J. Guitart, T. Victor, A. Moonsammy, S. Phillips, D. West, I. Lawrence, H. Jiang and Estee Lauder Laboratories, Melville, New York, New York M. Kobayashi We tested the effect of various cyclic phosphodiesterase (PDE) inhibitors on type IV PDE isolated North-western University, Chicago, Illinois; University of Chicago, Chicago, Illinois; and the from HaCaT cells. Rolipram and Ro-20–1724 are specific inhibitiors of type IV phosphodiesterase Fujisawa Research Institute of America, Evanston, Illinois (PDE4). IBMX, caffeine, and pentoxifylline are nonselective PDE inhibitors and as expected were The pathophysiology of atopic dermatitis may involve many cell types including CD4(ϩ) T cells, all effective at inhibiting cAMP PDEs present in HaCaT cell. Cyclic IMP, and 4-deoxyadenosine Langerhan’s cells, mast cells, and eosinophils. as suggested by Hanafin, Langerhan’s cells may be 3’:5Ј cyclic monophosphate were also effective cAMP PDE inhibitors. The compounds tested in of particular importance in the progression of this disease. Topical corticosteroids and topical the PDE assay were also tested for clinical anti-irritant ability against the contact irritant cinnamic tacrolimus (FK506) are both clinically effective treatments of atopic dermatitis. In order to better aldehyde. All the test agents treated sites were compared to cinnamic aldehyde treated skin sites. understand possible differences in the mechanisms of these two therapies, we performed an The percentage inhibition of erytherma has been determined. Pentoxifylline was found to inhibit immunohistochemical study to define the effects of these two treatments in patients with atopic cinnamic aldehyde induced inflammation by 50% compared to untreated sites. Other more specific dermatitis. Twenty-three patients meeting the Hanifin and Rajka criteria for atopic dermatitis inhibitors of phosphodiesterase type, such as rolipram and RO 20–1724, were found to inhibit were enrolled in a double-blind trial comparing tacrolimus 0.1% ointment and triamcinolone the cinnamic aldehyde by 69% and 57%, respectively. Caffeine inhibited cinnamic aldehyde contact 0.1% ointment. Skin biopsies were taken from target treatment lesions at baseline, after a treatment urticuria by 45% at 1% dose. period of 3 wk, and after a 2 wk post-treatment period. Clinical responses to therapy were similar A direct correlation was observed between the PDE inhibition and the anti-irritancy potential. in both treatment arms. Seventeen immunological markers were tested. The dermal CD4(ϩ) the agents with the stronger PDE inhibition in vitro showed the strongest anti-irritancy. These infiltrate improved after treatment in a similar fashion in both groups. Likewise, the population results stresse the value of PDE inhibitors as anti-inflammatories in topical use. of basophils and eosinophils did not differ significantly between the treatment arms of the study. Interestingly, staining for CD1a and CD11a and b, markers for Langerhan’s cells and macrophages, showed a significantly greater decrease in the group treated with triamcinolone. This result suggests that while topical steroids can induce Langerhan’s migration from the epidermis, tacrolimus likely functions to inactivate Langerhan’s cells. This result is the first in vivo evidence to corroborate a growing body of in vitro data which suggest that Langerhan’s cells can be inactivated by tacrolimus. We conclude that, while topical steroids and topical tacrolimus might both be effective therapy for atopic dermatitis, the mechanism by which these treatments ‘‘turn-off’’ epidermal Langerhan’s cells and induce clinical responses may be substantially different.

789 790 Effects of Isotretinoin on Bone Density and Calcium Metabolism Overexpression of Catecholamines in Vitiligo: Cause or Epiphenomenon? Cause or Epiphen- S. Leachman, K. Insogna, L. Katz, A. Ellison and L. Milstone omenon Departments Dermatology, Internal Medicine and Diagnostic Imaging,Yale University School of G. Orecchia, M. L. Cucchi, P. Frattini and G. Santagostino Medicine and VA Connecticut, New Haven, Conneticut IRCCS San Matteo. Institute of Pharmacology, University of Pavia, Italy Retinoids are important treatment for severe skin disease and for the prevention and treatment of The ethiology of vitiligo is still debated; yet neural factors seem to play a pivotal role in its cutaneous and noncutaneous malignancies. There have been few prospective studies regarding the pathogenesis. to establish a possible relationship between vitiligo and the activity of peripheral effects of retinoids on bone density in humans, despite a significant literature of retinoid-induced monoaminergic systems during different stages of the disease, the plasma levels of catecholamines osteoporosis and bone fractures in animals. We prospectively studied bone density and calcium [noradrenaline (NA), adrenaline (A) and dopamine (DA)], of their precursor 3,4-dihydroxyphenylai- metabolism over 6 mo in 18 otherwise healthy males (ages 17–25) who received isotretinoin (1 anine (DOPA), and their metabolites [3methoxy-4-hydroxyphenyigiycol (MHPG), normet- mg per kg) for cystic acne and in a control group of 14 healthy male volunteers (ages 19–26). anephrine (NMN), metanephrine (MN) and homovanillic acid (HVA)], as well as levels of 5- Bone density at Ward’s triangle decreased 4.4% in the isotretinoin group and was unchanged in hydroxyindolacetic acid (5-HIAA), which is the major metabolite of serotonin, were evaluated the control group. The difference between groups was statistically significant (P ϭ 0.044). Density by HPLC-ED. Seventy patients of both sexes (mean age Ϯ SE 34.0 Ϯ 1.7) with generalized (n ϭ at the femoral neck of the treatment group also declined, but the differences between groups in 43), segmental (n ϭ 4) or acrofacial (n ϭ 23) vitiligo and 20 healthy subjects, matched for sex bone densities at the spine and femoral neck were not significant. Serum levels of calcium, vitamin and age (mean Ϯ SE 29.2 Ϯ 3.1 y), participated in this study. D, and parathyroid hormone were unaffected by treatment with isotretinoin. Loss of bone density All considered biochemical parameters were higher in patients than in controls; this difference occurring in the absence of measurable alterations in calcium metabolism is likely to result from was significant for NMN, HVA and 5-HIAA. In the patient sample, significant relationships were a direct effect of retinoids on bone. found between monoamine and their metabolite levels: NA and A versus MHPG or metanephrines, as well as DA versus HVA; dopaminergic and serotoninergic parameters were also intercorrelated. Patients with more recent onset (Ͻ1 y) showed significantly increased A, NA, DA and metanephrines levels in comparison to those of patients with vitiligo for a longer period (Ͼ10 y). Few significant differences were noted in patients subdivided according to vitiligo type, and none according to age of disease onset. These data seem indicate a stimulate catecholamine turnover due to stressful events, among them the onset of vitiligo itself. Whether the overexpression of catecholamines and metabolites in the plasma of vitiliginous patients may be the principal cause of depigmentation or simply an epiphenomenon has to be determine yet.

791 792 Vascular Hyporeactivity in Perniosis and Acrocyanosis Broad Specificity Neutral Proteases Reduce Visual Scaling S. Datta, S. Chopra, H. Bull and P. Dowd K. El-Kadi, C. Feinberg, D. Pocalyko, C. Nunn, A. Battaglia, P. Chandar, N. Richardson and UCLMS, London, UK R. Sabin Perniosis (P) and Acrocyanosis (A) are cold related disorders of unknown pathogenesis. Recently Unilever Research US, Edgewater, New Jersey immunohistochemistry (IHC) and in vivo pharmacological (I-VP) studies have demonstrated a For effective desquamation to occur, desmosomes must undergo proteolytic hydrolysis. In aberrant deficit in sensory-motor vasodilator peptidergic Calcitonin Gene Related Peptide (CGRP) desquamatory conditions such as xerosis, this process is impeded, resulting in an excessive containing neurones in primary Raynaud’s phenomenon (1°RP), RP with Systemic Sclerosis (SS) accumulation of corneocytes on the skin surface. Exogenous proteases were examined in this and RP associated with occupational exposure to vibration [vibration white finger (VWF)]. In research for their ability to reduce the scaling associated with soap induced dryness. Randomized, VWF, IHC using the pan neuronal marker [antibody to protein gene product 9.5 (PGP 9.5)] double-blind paired comparison studies were conducted in which subjects were treated with a demonstrated a more profound neuronal deficit in keeping with the persistent painful paraesthesia formulation containing a protease in an aqueous solution followed by a moisturizer on one outer of that condition. I-VP findings were also consistent with this. In this study IHC and I-VP were lower leg and an aqueous buffered solution vehicle control followed by a moisturizer on the other employed in an attempt to define neurovascular and/or vascular abnormalities of putative leg. the studies were 15 d in duration, consisting of (i) a seven day soap induced dry-down phase, pathogenic importance in P and A. Ethical approval was obtained and all subjects gave informed (ii) a 4 d product application phase, and (iii) a 3 d soap challenge. Visual assessments of dryness consent. Biopsies of dorsal digital skin were obtained under lignocaine LA from patients with P and erythema were conducted using a nine-point scale at baseline, throughout the product andA(nϭ10) and normal volunteer age, sex and smoking matched controls (n ϭ 10) and application phase and after the 3-d soap challenge. The results showed that proteases produced by processed for IHC which was performed with specific antisera for PGP 9.5, CGRP and von Bacillus or Aspergillus gave significantly greater reduction in visual dryness from baseline compared Willebrand factor (vascular endothelial cell marker) using a DAB-nickel technique. to vehicle. Greater dryness reduction was also achieved by proteases from Bacillus or Aspergillus Qualitative and quantitative analysis (by computerised image analysis) was performed on the blood compared to proteases with more narrow substrate specificity such as papain, and proteases with vessels and neurones in the epidermis and dermis of multiple sections from each biopsy (10 µm a pH optimum less than pH 5 (e.g. acid fungal protease). Additionally, Alcalase and Optimase, both sections). Intradermal injections of 25 µl of Histamine 10 µg per ml (Hi), endothelin-1 2 µM produced by Bacillus licheniformis, showed the expected dose response, with higher concentrations of (ET-1) and CGRP (1 µM) were made into the middle phalangeal dorsal digital skin at 21°C and enzyme giving a greater reduction in mean dryness scores. There was no difference in performance 10°C in an environmental chamber. Cutaneous blood flow was measured by Laser Doppler between Optimase and Alcalase in cells with matched caseinolytic activities. Microbial proteases Flowmetry (LDF) prior to and at 2, 10 and 30 min after injection. The areas of pallor and/or with broad substrate specificity and a pH optimum between pH 5–9 were most weight efficient. erythema were calculated by planimetry. Statistical analysis of results of quantitative IHC and I- VP was by unpaired 2 tailed Student’s T-test and by a Mann Whitney U nonparametric test. The IHC was qualitatively and quantitatively normal in P ϩ A but ET-1 and Hi flares were significantly (P Ͻ 0.05) decreased at 10°C (ET-1 flare 2 min) (Hi flare: at 2 & 30 min). Patients also exhibited significantly (P Ͻ 0.05) decreased blood flow (LDF velocity) at 10°C Hi flare (2 min), ET-1 flare (2 min and 10 min), and CGRP erythema (30 min). These findings indicating a generalised vascular hypo-reactivity are in contrast to those defining a neuronal deficit in RP, SS and VWF and suggests an underlying vascular as opposed to neurovascular pathogenic mechanism in P ϩ A. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 655

793 794 Corticosteroid (Clobetasol Propionate) Induced Skin Atrophy Analyzed with Non-invasive Randomized, Controlled Trial of Narrow Band UVB (TL01) and UVA in Adult Atopic Dermatitis Methods N. Reynolds, V. Franklin, J. Gray, B. Diffey and P. Farr L. Kolbe, V. Schreiner, T. Stoudemayer and A. Kligman Department of Dermatology, University of Newcastle upon Tyne; Centre for Health and Medical Paul Gerson Unna Research Center, Beiersdorf AG, Hamburg, Germany; SKIN Inc., Consho- Research, University of Teesside and Regional Medical Physics Department, Newcastle General hocken, Pennsylvania; Department of Dermatology, University of Pennsylvania, Philadelphia, Hospital, UK Pennsylvania Previous studies have suggested that ultraviolet (UV) B and UVA, either singly or in combination Atrophy is one of the most adverse side-effects of topical steroid treatment. For the development may be effective treatments for atopic dermatitis (AD). However, the majority of previous studies of novel steroids it would be useful to have reliable noninvasive methods to monitor the have been conducted on patients with relatively mild AD and/or have not been blinded. We atrophogenicity of steroid formulations. wished to determine whether UV sources, that would be widely available to dermatologists, We treated subjects on the volar forearm with clobetasol propionate (Temovate cream 0.05%) for provide effective adjunctive treatment for patients with moderate/severe AD. Seventy-three adult 4 wk and monitored skin changes. (i) Ultrasound for skin thickness in vivo; (ii) confocal laser patients with AD were randomized in balanced blocks to receive either narrow band UVB (TL01, microscopy for epidermal changes; (iii) detergent scrubs for corneocyte analysis; (iv) evaporimeter up to a maximum of 1.2 J per cm2), UVA (TL09, up to a maximum of 15 J per cm2) or placebo for TWL; (v) DMSO for penetration; (vi) sodium hydroxide erosion assay for barrier integrity; (visible-light fluorescent lamps) phototherapy twice weekly for 12 wk. Patients continued topical (vii) cyanoacrylate biopsies for ceramide analysis treatment with mild/moderate steroids and emollients during the trial. Clinical assessments were Ultrasound showed thinning of the skin within 1 wk of treatment; confocal laser microscopy made by a single observer, who was unaware of treatment allocation, at baseline and after every revealed a thinning and flattening of the epidermis and a decrease in keratinocyte size. Corneocyte six treatments. The primary outcome measures were designated as disease activity (five clinical analysis showed a decrease of corneocyte size. TWL and DMSO reaction increased during features at six separate body sites) and extent of disease, according to the rule of nines. Data were treatment, whereas erosion times were decreased, indicating an impaired barrier. Lipid content of analysed by the method of summary measures in which a regression slope is fitted for each patient the stratum corneum decreased with treatment. to the dependent variable over time. Data were included for analysis if the patient attended for at least two follow-up assessments. Sixty-nine patients commenced therapy, 22 patients withdrew and 47 patients completed treatment. The treatment groups were well matched with respect to baseline assessments and number of withdrawals. Over 24 treatments with TL01 phototherapy, total disease activity and extent of disease reduced on average by 29% (95% CI 42%–17%) and 9% (95% CI 14%–5%), respectively (n ϭ 22). Treatment with TL01 reduced total disease activity on average by 9.4 points (95% CI 15.2–3.6 points) more than placebo and reduced extent of disease on average by 4.9% (95% CI 11.9% to 1.5%) more than placebo. No significant changes were observed following phototherapy with UVA (n ϭ 19) compared to placebo (n ϭ 19). This study demonstrates the efficacy of TL01 phototherapy in the treatment of moderate/severe adult AD but does not provide support for the effectiveness of UVA.

795 796 Paclitaxel: Anti-Angiogenic Effects In Vitro and Inhibition of Experimental Skin Inflammation Immunosuppresive Activity of Spirogermanium (SG) on T-Cell Activation and Inflammatory In Vivo Cytokine Secretion In Vitro P. Velasco, L. Richard, W. Hunter,* P. Toleikis* and M. Detmar A. Abdulghani, S. Rose, L. Bernstein* and A. Gottlieb Cutaneous Biology Research Center, Department of Dermatology, Massachusetts General Hospital Division of Clinical Pharmacology, Clinical Research Center, UMDNJ -Robert Wood Johnson and Harvard Medical School, Boston, Massachusetts; *Angiotech Pharmaceuticals Inc., Vancouver, Medical School, New Brunswick, New Jersey; and *GeoMed, Inc., Menlo Park, California British Columbia Spirogermanium (SG) is a novel germanium-containing azasprine compound reported to have Paclitaxel is widely used as a potent anticancer agent. Because of evidence for an antiangiogenic antitumor, antiarthritic, and immunomodulatory activity. in this study we were interested in activity of paclitaxel, we investigated the effects of paclitaxel on human dermal microvascular determining the immunosuppressive potentials of SG. We isolated peripheral blood mononuclear endothelial cells (HDMEC) in vitro. Moreover, we studied the effects of a topical paclitaxel cells (PBMC) from healthy human volunteers, induced lymphocyte proliferation with phytohemag- formulation on skin inflammation induced by PMA and on oxazolone-induced cutaneous glutinin, and incubated them with SG in vitro from concentrations of 0.0006 µg per ml to 10 µg hypersensitivity reactions. per ml in vitro. After an incubation period of 72 h, cell viability was determined using trypan blue Paclitaxel dose-dependently inhibited cell growth and DNA synthesis of proliferating HDMEC dye exclusion method, a cell proliferation assay was done using BrdU incorporation assay, and cultures. In contrast, little or no effects on HDMEC growth were observed in confluent cultures interleukin-2 (IL-2), gamma interferon (01γ-IF) and tumor necrosis factor alpha (TNF-01α) characterized by low proliferative activity. Topical application of PMA to the ear skin of 8-wk- cytokine enzyme-linked immunosorbent assays (ELISA) were done on cell culture supernatants. old FVB mice induced a strong inflammatory reaction with pronounced ear swelling, angiogenesis, SG was shown to effectively inhibit T-lymphocyte proliferation in vitro by around 100% at and leukocyte infiltration. When a 1% paclitaxel formulation was applied topically immediately concentrations 0.0012–0.15 µg per ml at which cell viability was found to be above 95%. IL-2 before or shortly after PMA application, and thereafter once daily, a potent inhibition of ear and 01γ-IF secretions were strongly inhibited at concentrations of SG above 0.0012 µg per ml, swelling, redness, angiogenesis, and inflammatory infiltration was observed. In contrast, no TNF-a secretion was strongly inhibited at concentrations above 0.0098 µg per ml. Our results detectable effects were found after vehicle application, and application of paclitaxel to normal skin demonstrate that SG is a potent inhibitor of mitogen induced T cell proliferation and the secretion did not induce any detectable skin irritation. Sensitization with 3% oxazolone in acetone:olive oil of inflammatory cytokines IL-2, 01γ-IF, and TNF-01α at noncytotoxic concentrations. SG may (4:1; vol:vol), followed by re-challenge with 1% oxazolone to the ear skin after 5 d reliably have potential beneficial effects in T cell mediated-disorders such as psoriasis, rheumatoid arthritis, induced contact dermatitis with pronounced edema. Topical application of paclitaxel, but not inflammatory bowel disease, multiple sclerosis and transplantation rejection. vehicle, significantly reduced the extent of ear swelling. These findings demonstrate antiangiogenic properties of paclitaxel in vitro and in vivo, and suggest that paclitaxel, besides its anticancer activity, might be beneficial in the treatment of inflammatory skin diseases which are characterized by angiogenesis.

797 798 Ultraviolet B Induction of Cytochromes P 450 1 A1 and 1B1 in Human Skin Generic Tretinoin Gels: Demonstration of Bioequivalence by Pharmacodynamic and Pharmaco- S. K. Katiyar, M. S. Matsui,* K. Marenus* and H. Mukhtar kinetic Assays Department of Dermatology, Case Western Reserve University, Cleveland, Ohio, and 01*Estee P. Lehman, N. Agrawal, K. Spear* and T. Franz Lauder Research Laboratory, Melville, New York University of Arkansas for Medical Sciences, Little Rock, Arkansas; *Spear Pharmaceutical, Fort Cytochromes P 450 (CYPs) belong to a multigene family of heme proteins which catalyze the Myers, Florida oxidation of various xenobiotics and endogenous molecules. The expression of CYPs within The objective of this study was to demonstrate the bioequivalence of two generic tretinoin gels target cells of human skin is an important determinant of susceptibility to adverse effects of some (0.01% and 0.025%) to the innovator products, Retin-A Gel. small molecular weight compounds that cause cutaneous allergic reactions, cancers and other Bioequivalence was assessed using two model systems: (i) In Vitro Cadaver Skin Permeation (CSP). diseases. Two members of CYP1 family CYP 1A1 and 1B1 are highly active in the bioactivation All four products were spiked with 3H-tretinoin and applied to split-thickness cadaver skin. the of aromatic amines and ubiquitous environmental pollutants such as polycyclic aromatic hydrocar- tretinoin absorption was measured by multiple sampling of the dermal receptor solution over 48 bons to which the skin is exposed. The effect of solar ultraviolet (UV) B exposure to CYPs in h and assaying for radioactivity. (ii) In Vivo Transepidermal Water Loss (TEWL). Three 5 cm2 sites human skin is not known. In this study the effect of UVB exposure to environmentally protected (placebo included) on both forearms of 36 volunteer subjects were dosed with 4 mg per cm2 of buttock skin on CYP 1A1 and 1B1 was determined using immunohistochemistry, RT/PCR and product for 21 d. For the first 5 h, the sites were occluded. Daily readings of TEWL (by western blot analysis. Using immuno-histochemistry, in non-UVB exposed skin, we found that Evaporimeter) and scaling (0–3 visual scale) were made and used as the basis to determine CYP 1A1 was mainly localized in basal cell layer of the epidermis while CYP 1B1 was localized bioequivalence. in other than the basal cells of the epidermis. UVB exposure to skin resulted in a dose-dependent ANOVA with 90% confidence intervals were used to test for bioequivalence. (i) CSP Total tretinoin (1–4 MED) and time-dependent (6–48 h) induction of both CYP isozymes in the epidermis. absorption from the generic gels was 106.3% and 95.5% of the innovator products for the 0.01% RT/PCR and western blot analyses revealed that UVB exposure (4 MED) resulted in enhanced and 0.025% strengths, respectively. (ii) TEWL the generic gels demonstrated 104.5% and 109.4% expression of mRNA and protein of both CYP 1A1 and 1B1. Smoking status did not appear to peak TEWL response, and, 99.7% and 100.7% scaling response to 0.01% Retin-A and 0.025% affect the ability of UVB to induce CYP 1A1 or 1B1. UVB induction of CYPs in human skin Retin-A, respectively. may result in enhanced bioactivation of other environmental pollutants to which the humans Both CSP and TEWL assays demonstrated bioequivalence of the corresponding strengths of the are exposed. generic tretinoin gel products well within the conventional 80% to 125% confidence interval acceptance range. These methods were validated by the demonstration of the nonbioequivalence between the low and high strengths of the tretinoin gel products. 656 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

799 800 Human Wound Healing Models for Assessing Safety and Efficacy of Topically Applied Skin SDZ ASD 732, a Novel Ascomycin Derivative with Local Anti-Inflammatory Activity, Lacks Care Products Systemic Immunosuppression T. Stoudemayer, A. Kligman, L. DeShields, M. Stoudemayer, R. Mass-Irslinger and O. Mills P. Ulrich, J. M. Alonso, W. Suter, A. Cordier, R. Ettlin, M. Grassberger and J. Meingassner SKIN Inc., Conshohocken, PA, Beiersdorf, Inc., Norwalk, Conneticut; and Foundation for Basic Preclinical Safety, Novartis Pharma AG, Basel, Switzerland; Novartis Research Institute, Vienna, Cutaneous Research, Philadelphia, Pennsylvania Austria A great variety of emollient formulations are available for treating skin which has been traumatized SDZ ASD 732 is an anti-inflammatory ascomycin derivative which does not bind to macrophilin by cuts, abrasions, sunburn, chemical irritation, etc. Surprisingly there have been very few studies in vitro and has no activity in models indicative for systemic immunosuppression in vivo.To which have unequivocably demonstrated that these products promote wound healing or indeed, establish a direct relationship between the anti-inflammatory activity following topical application whether they impair the reparative process. We have developed four models to investigate how of SDZ ASD 732 and it’s effect on the skin-draining lymph node (LN), we used the murine emollients affect the healing of experimental wounds. (i) Three MED’S from a Solar Simulator; Local Lymph Node Assay (LLNA) as an in vivo model for contact allergy (CA). In this study the (ii) Chemical injury from an overnight occlusive application of 1% sodium lauryl sulfate; (iii) influence of SDZ ASD 732 on the oxazolone-induced changes in the ear-draining LNs and in traumatic removal of the horny layer by 50 Scotch Tape strips; (iv) 1% canthardin blister wound. the ear tissue during the challenge phase of CA was investigated and directly compared with the The Test emollients were applied daily for 1–2 wk Assessments include: (i) transepidermal water effects of FK 506, a potent immunosuppressant with high affinity to macrophilin. Groups of mice loss; (ii) conductance; (iii) Laser-Doppler Velocimetry; (iv) colorimetry (Minolta); (v) reflection received oxazolone topically on the shaved back to induce contact allergy. After a recovery period spectrophotometry; (vi) polarized digital photography; (vii) Silflo replicas; (viii) D’Squames; (ix) of 14 d mice were challenged once with oxazolone on the dorsum of both ears. Topical application clinical evaluations. We looked at hydrophilic ointment (Petrolatum), water in oil emollient of SDZ ASD 732 and FK 506 were performed on four consecutive days starting 1 d before the (Aquaphor Healing Ointment), oil in water emollient (Nivea Cream), and USP Cold Cream. In single challenge. Auricular LNs draining the ear tissue were excised 24 h following the last relation to the untreated control these agents exerted a beneficial effect in repair. However, it was exposure. Evaluation consisted of assessing LN weights to monitor organ hyperplasia and Interleukin possible to arrange each item in a rank order of efficacy. (IL)-2 receptor expression to assess the immune cell activation state following substance application. Furthermore, cytokine release (IL-2, IL-4, IL-6, IL-12 and IFN01γ) by lymph node CD4 T cells and antigen-presenting cells restimulated in vitro was investigated. Measurement of ear swelling was performed 24 h before and 5 h, 24 h and 48 h after the challenge. Direct comparison of SDZ ASD 732 and FK 506 at concentrations of 0.1% (wt/vol) showed that FK 506 was a potent inhibitor of all oxazolone-induced changes (increased LN weights, IL-2R expression, release of IL-2/4/6/10/12 and IFN01γ) in the ear-draining LNs, whereas SDZ ASD 732 showed no activity. However, SDZ ASD 732 was nearly as effective as FK 506 in the inhibition of challenge-induced ear-swelling indicating its anti-inflammatory activity in the treated skin without affecting the related lymphoid organs. In conclusion, SDZ ASD 732 represents a potential novel therapeutic agent for inflammatory skin diseases, which lacks systemic immunosuppressive activity.

801 802 Sunscreens with High SPF Values are Not Equivalent in the Protection from UVA Induced The Generation of Reactive Oxygen from Urocanic Acid in the UV-A Polymorphous Light Eruption J. Simon H. Stege, M. Budde, A. Richard,* A. Rougier,* T. Ruzicka and J. Krutmann Department of Chemistry, Duke University, Durham, North Carolina Clinical and Experimental Photodermatology, Department of Dermatology, University of Dussel- Inspectroscopic methods are used to model the in vivo photoreactivity of the proposed immunomod- dorf, Germany; *La Roche-Posay Pharmaceutical Laboratories, La Roche-Posay and Cour- ulatory epidermal chromophore trans-urocanic acid. The data show that trans-urocanic acid sensitizes bevoie, France the reactive oxygen species singlet oxygen in two distinct regions of its absorption spectrum: near Polymorphous light eruption (PLE) represents an abnormal response of human skin to ultraviolet the peak of the absorption spectrum at 270 nm and in the weakly absorbing UV-A region between (UV) radiations, which is characterized by increased and prolonged expression of proinflammatory 320 nm and 351 nm. An UV-A action spectrum for singlet oxygen production by trans-urocanic molecules such as ICAM-1 on the surface of keratinocytes (KC). Photoprovocation studies have acid will be presented and compared to UV-A-induced photoaging effects reported in the literature. previously revealed that in vast majority of PLE patients (80%) skin lesions can be induced through The data reflect that a skin chromophore must be studied throughout its entire absorption profile repetitive exposure to longwave UVA radiation (UVA 1, 340–400 nm). to gain a complete understanding of the molecule’s physiological effects. This work was supported In the present study we have assessed whether the generation of PLE lesions can be effectively by NIGMS. prevented through pretreatment of human skin with three different sunscreen formulations having the following protection factors: Sunscreen A: SPF Ͼ 75; UVA-PF 15 (IPD: Imediat Pigment Darkening method). Sunscreen B: SPF ϭ 35; UVA-PF (not known). Sunscreen C: SPF Ͼ 60; UVA-PF 28 (PPD: Persistent Pigment Darkening method). In this double blind, intraindividual comparative study, nine patients with UVA PLE were photoprovocated by exposing four sensitive skin areas to 100 J per cm2 of UVA 1 on 3 consecutive days. Prior to photoprovocation, skin areas were either left untreated, or pretreated with crean A, BorC. We have found that cream C was highly effective in providing complete protection against UVA radiation-induced skin lesions in nine of nine patients. In contrast cream A provided partial protection in seven of nine and complete protection in only two of nine, and cream B protected partially in one of nine and completely in none of nine patients, whereas eight of nine showed no protection. The very high protective effect of cream C was corroborated by immunohistochemical studies in which strong KC ICAM-1 expression was found in unprotected skin areas, but not in cream C pretreated areas. These studies clearly show that formulations having similar SPF values are not equivalent in preventing from UVA-induced PLE and that there is a need for products covering the entire UV spectrum, UVB ϩ UVA. Moreover, we, for the first time, demonstrate the efficacy of a novel UVA filter (Mexoryl XL)

803 804 The Size of Dependent Optical Properties of Melanins Down-Regulation of CXCR2 but Not CXCR1 Expression in Human Keratinocytes by UVB J. Simon, S. Forest and J. Nofsinger S. Kondo, A. Yoneda, H. Yazawa and K. Jimbow Department of Chemistry, Duke University, Durham, North Carolina Department of Dermatology, Faculty of Medicine, Sapporo Medical University, Sapporo, Japan The importance of melanin as a photoprotection and/or photosensitization agent in human skin Chemokine activation of target cells is mediated by two specific receptors: CXCR1 and CXCR2. is not well understood. The resolution of these issues is directly linked to an understanding of the Both receptors bind IL-8 with high affinity but have different affinities for MGSA and NAP-2. origin of the pigments optical properties and their associated photoreactions. In this presentation, It has been shown that the expression of epidermal CXCR2 is increased in psoriasis and suggested the absorption and emission properties of eumelanin (from Sepia Officinalis/) are examined as a that activation of KC mediated by CXCR2 contributes to the characteristic epidermal changes function of the size of the aggragate. We find that the absorption and emission properties of the observed in psoriasis. In order to examine the mechanism(s) by which UVB therapy is effective chromophore depend strongly on aggragate size. Specifically, with decreasing particle size, for several dermatoses including psoriasis, we sought to examine if UVB would modulate the eumelanin becomes a significantly poorer absorber in the UV-A. Correspondingly, with decreasing expression of CXCR1 and CXCR2 in human KC. Constitutive expression of CXCR1 and particle size, the emission spectrum shifts to higher energy. These data have implications about CXCR2 mRNA was detected by RT-PCR in normal cultured human KC. After 100 or 300 J the possible photoprotective role of eumelanins. In addition, photoacoustic measurements enable per m2 irradiation, a decrease in CXCR2 mRNA was detectable from 6 h after irradiation and us to conclude that sacttering accounts for at most 15% of the spectral response observed in the this downregulation was observed until 48 h after irradiation. By contrast, CXCR1 mRNA level UV-B. was unchanged. Immunohistochemical studies on 2MED exposed and unexposed skins from normal volunteers revealed that CXCR2 staining was localized to the suprabasal layer of the epidermis but 24 h after 2MED irradiation, CXCR2 staining was decreased. A faint CXCR1 staining was observed in the lower part of the epidermis both in unexposed and exposed skins. Our results suggest a possible role for the IL-8-CXCR2 signaling pathway in the skin after exposure to UVB, and that UVB-induced growth inhibition of KC in hyperproliferative skin disorders may be related to downregulation of CXCR2. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 657

805 806 PIK-Related Kinases in the Response to DNA Damage by UV Endogenous Antioxidant Enzyme Activities in Human Skin are Decreased by Four but not One P. Nghiem, B. Desai and S. L. Schreiber Exposure to Ultraviolet Light Howard Hughes Medical Institute; Department of Chemistry and Chemical Biology, Harvard E. A. Duell, S. Kang and J. J. Voorhees University, Cambridge, Massachusetts; and Center for Cutaneous Oncology, Dana-Farber Cancer Department of Dermatology, University of Michigan Medical School, Ann Arbor, Michigan Institute, Boston, Massachusetts The utilization of oxygen to sustain life produces low levels of reactive oxygen species (ROS) The protective response of cells to DNA damage by ultraviolet radiation involves cell cycle arrest which damage proteins, lipids and nucleic acids. Exposure of skin to ultraviolet (UV) light and induction of DNA repair enzymes. This response is dependent on functional p53 and involves generates additional ROS in skin. We investigated the effect of one 2 minimal erythema dose the induction of p21, an inhibitor of cell cycle progression. The protein which senses UV-induced (MED) or four 1 MED UV exposures on endogenous antioxidant enzyme activities which convert DNA damage and activates p53 is not known. The response to DNA damage in yeast and ROS to less damaging compounds. A UVA ϩ B source with a 320-nm filter was used to mimic mammals is mediated by members of the phosphatidyl inositol kinase (PIK)-related kinase family. solar irradiation. Biopsies obtained 20 min after the final UV exposure were extracted and analyzed There are four known PIK-related kinases in humans: (i) the gene which is mutated in ataxia- for antioxidant content as well as antioxidant enzyme activities. Activities that showed a statistically telangiectasia (ATM) necessary for the ionizing DNA damage response but not the UV response, significant decrease (P Ͻ 0.05, n ϭ 12) after four 1 MED exposures were catalase (48%01↓), (ii) the SCID mouse gene, DNA-PK, necessary for proper antigen receptor VD-J recombination superoxide dismutase (40%01↓), GSH peroxidase (20%01↓), and GSH reductase (33%01↓). but not the UV response, (iii) the target of the immunosuppressant rapamycin (FRAP) responsible However, there were no significant changes in GSH transferase and thioredoxin reductase activities. for G1/S arrest following nutrient or mitogen deprivation, and (iv) ATR (ataxia-telangiectasia These same enzyme activities in biopsies obtained after one 2 MED exposure were not significantly related), which is most closely related to Mec-1 which mediates the UV response in S. Cerevisiae. different (P Ͼ 0.05, n ϭ 6) from activities in unirradiated areas. Endogenous antioxidants such as One of these PIK-related kinases is likely to be the mediator of the UV response. A possible role reduced glutathione, vitamin C, and thioredoxin were not significantly altered after exposure to for FRAP had not been tested in the UV response. Using rapamycin (10 nM) to block FRAP four 1 MED or one 2 MED doses of UV. Only endogenous 01γ-tocopherol but not 01α- function, we found that UV-induction of p53 and p21 proceed normally in primary human tocopherol showed a significant 27%01↓ (P ϭ 0.05) after four 1 MED exposures. These data fibroblasts, strongly suggesting that FRAP is not necessary for the UV response. Based on data show that the endogenous antioxidant enzyme system in human skin can withstand one 2 MED from several groups it appears that ATR is a good candidate for mediating this UV pathway. exposure but not four 1 MED exposures. Such analyses can be used to design topical antioxidant Efforts to study ATR’s role in the activation of p53 by UV have been hampered by the fact that combinations at concentrations sufficient to provide verifiable preservation of the endogenous overexpressing or inhibiting ATR function is lethal to cells with wild type p53. We are currently antioxidant enzyme system. generating a system to assess in vitro induction of specific DNA binding by p53 and the role of this PIK-related kinase in the response to DNA damage by UV.

807 808 A Progress Report on Narrowband Ultraviolet B Phototherapy P53-Dependent DNA Damage Response after Ultraviolet B Irradation in Skin Cells – A Useful J. J. Kim and H. W. Lim Indicator for UV-Induced Cellular Stress and DNA Damage Department of Dermatology, Henry Ford Health System, Detroit, Michigan K. Will, M. Neben, T. Schmidt-Rose, K-P. Wittern, W. Deppert and J. Bergemann Narrowband ultraviolet B (UVB) phototherapy is a relatively new treatment modality in the field Paul Gerson Unna Research Center, Beiersdorf AG, Hamburg, Germany, Heinrich-Pette-Institut, of photodermatology in the United States. There is a large body of evidence supporting the Hamburg, Germany efficacy and superiority of narrowband UVB therapy over other forms of phototherapy in the A strong association has been found between exposure to UV radiation, DNA damage associated management of photoresponsive dermatoses. However, a consensus has yet to be reached regarding changes of skin cells, and a higher risk for tumor development. To prevent permanent changes in an optimal treatment protocol. We report our progress with a protocol for narrowband UVB the genome, damaged cells activate checkpoint control elements to arrest cell cycle progression therapy that appears to be effective for a variety of photoresponsive dermatoses. A retrospective and facilitate DNA repair. Elevation of the tumour suppressor p53 expression level even after study was done on 24 patients who received narrowband UVB therapy. Indications for therapy exposure to low doses of UV light is one of the cellular answers of skin cells to UV stress. included psoriasis vulgaris (n ϭ 11), vitiligo (n ϭ 4), pityriasis lichenoides chronica (n ϭ 1), Therefore this reaction can be used as a sensitive detector of genotoxic cell stress. Effective prurigo nodularis (n ϭ 2), lichen planus (n ϭ 1), mycosis fungoides (n ϭ 2), pruritus secondary sunscreen products prevent UV-induced upregulation of p53. Thus quantification of p53 expression to chronic renal failure (n ϭ 2), and dermatitis (n ϭ 1). Skin types ranged from Fitzpatrick type after UV radiation can be used to evaluate the efficiency of UV protection by sunscreen products. I to VI. Treatment was given three times per week. Starting doses were 70% of the minimal Since the basal expression level of p53 as well as the UV damage response in keratinocytes can erythema dose (MED), with dose increments of 10%–15% if no erythema was present, and 0% if be influenced also by cell–cell interactions and cellular adhesion we optimized the growth moderate erythema was present. Treatment was temporarily suspended if severe erythema conditions with respect to UV reactivity. Cocultivation of fibroblasts and keratinocytes resulted developed. Patients were assessed regularly to score disease severity and monitor adverse events. in a decrease of the basal p53 level of the keratinocytes and an increase in the UVB-induced p53 Eighteen of the 24 patients remained in therapy at the time of the review. Only one of the six response of the keratinocytes. Additionally we observed that different types of skin cells can patients discontinued therapy because of a paradoxical sunburn reaction to this and subsequent mutually influence their reactivity in response to UV irradiation. Furthermore using these culture forms of light therapy. Only two of the 18 patients experienced moderate to severe erythema that conditions UV-induced elevation of p53 was significantly decreased by applying sunscreen products. necessitated withholding treatment or covering affected areas with sunblock. There was an overall decrease in disease severity scores for all skin conditions, except in the one case of lichen planus which showed no visible improvement. Our experience with narrowband UVB therapy as an effective form of phototherapy is consistent with previous reports. We propose that a starting dose of 70% of the MED with subsequent dose increments of 15%/0% results in good treatment outcomes with an extremely low incidence of burning.

809 810 Role of Fas Associated Death Domain Protein in UVB Induced Apoptosis p53 Mutations at Psoralen Photochemical Hot Spots – Rare but Significant Events in PUVA L. Zhuang, B. Wang, W. Yeh,* G. Shivji, T. Mak* and D. Sauder Induced Skin Cancers Department of Dermatology, Sunnybrook Health Science Centre, and *Amgen Institute, Depart- Z-P. Ren, J. McNiff* and F. P. Gasparro† ment of Medical Biophysics and Immunology, University of Toronto, Toronto, Canada Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, New York; Apoptosis has been recognized as an important control mechanism in both the maintenance of *Department of Dermatology, Yale University, New Haven, Conneticut; and †Department of tissue homeostasis and elimination of cells with damaged DNA. UVB-induced apoptosis has Dermatology Thomas Jefferson University, Philadelphia, Pennsylvania become a subject of interest, due to the potential link between this process and UVB-induced To better understand the development of squamous cell carcinoma (SCC) in psoralen/UVA carcinogenesis. However, the molecular mechanisms for UVB-induced cell apoptosis have not (PUVA) treated psoriasis patients, genetic alterations were examined in the p53 gene. Preliminary been fully elucidated. FADD (Fas-associated death domain protein) is a signal transducer acting results on the nature of p53 mutations in human specimens indicated there were significant downstream of the cell death receptor Fas and TNF-Rp55. FADD signaling activates a cysteine differences compared to in vitro cellular and in vivo animal studies. Fourteen biopsies from four protease cascade leading to cell death. The role of FADD in UV-induced apoptosis was examined additional PUVA treated psoriasis patients were analyzed including one patient from whom biopsies using FADD-deficient cultured embryonic fibroblasts (EFs). Both FADDϩ/ϩ and FADD–/– EFs from four separate lesions were obtained. A microdissection technique was used to dissect the were irradiated with UVB, and the degree of apoptosis was determined by flow cytometry, in situ normal and tumor cells within the same biopsy. DNA extraction, PCR and direct sequencing of nick end labelling of DNA and a DNA ladder assay. Our finding revealed that UV-induced p53 gene (exons 5–8) and immunohistochemistry were performed. Nine mutations were found. apoptosis in EFs in a dose-dependent and time-dependent manner. Deletion of FADD in EFs One patient showed seven mutations in four separate lesions. Each of the four different lesions significantly suppressed UV-induced apoptosis. These results indicate that ablation of FADD in showed a PUVA-type T01→G transition at codon 205. This mutation occurred within a DNA EFs results in suppression of UVB-induced apoptosis. sequence containing a likely spot for psoralen photochemistry (GAG TAT TTG GAT). In addition three CG01→TA transitions typical of solar exposure were also observed. Another mutation was in stop codon which terminated the p53 protein synthesis. In an actinic keratosis from another patient calf lesion, another CG01→TA transition was found. The genetic alterations were correlated with p53 expression measured by immunohistochemistry. These results clearly implicate photoinduced damage (either solar or PUVA) as an important factor in the development of these lesions. However, the lack of an overwhelming preponderance of PUVA type mutations suggest that the PUVA SCC may arise by more than one mechanism. PUVA mutations may lead directly to cellular transformations. Alternatively, PUVA could induce skin immune suppression that allows transformed cells harboring previously induced solar p53 mutations to develop into frank tumors. 658 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

811 812 Direct and Indirect Effects of Ultraviolet Radiation on Collagen Synthesis by Fibroblasts in Reduction of Laser Thermal Damage Using a Novel Device and the Free-Electron Laser Collagen Gels N. C. Spector, L. Reinisch and D. L. Ellis S. Kossodo, G. Simon and I. E. Kochevar Department of Medicine, Division of Dermatology, Vanderbilt University and Nashville Veterans Wellman Laboratories of Photomedicine, Department of Dermatology, Massachusetts General Administration Medical Centers, Nashville, Tennessee; and Department of Otolaryngology, Hospital, Harvard Medical School, Boston, Massachusetts Vanderbilt University, Nashville, Tennessee Chronic exposure to ultraviolet radiation (UVR) results in premature skin aging or photoaging, We investigated heat-conducting templates as a method of reducing laser induced lateral thermal characterized by degeneration of collagen fibers and modification of the ratios between different damage at various infrared wavelengths. Partial thickness laser incisions were made on in vitro types of collagen. UVR can modulate collagen expression, either directly by acting on cells human skin with a computer controlled standardized delivery of infrared radiation using the Free- secreting it or indirectly by inducing skin cells to secrete mediators which influence collagen Electron Laser (FEL) at wavelengths of 3.0, 6.45, and 7.7 microns, with and without an aluminum expression. We investigated the synthesis of collagen by fibroblasts in three-dimensional collagen heat conducting template. Additionally, we studied the effects of different conductivity templates gels and compared the results to those obtained in monolayer cultures. Normal human foreskin using copper, aluminum, glass, and plexiglass at 7.7 microns. All of the skin samples (N Ͼ 50) dermis fibroblasts were seeded into collagen type I/III gels, or as monolayers. At different times were analyzed for lateral cutaneous thermal damage using computerized morphometric analysis. during the 10-d culture period, cells were tested for their capacity to synthesize collagen, by the The aluminum template lowered the mean lateral thermal damage 83% at 3.0 microns (P Ͻ incorporation of radiolabeled proline. Cells in gels produced significantly less collagen than cells 0.007), 45.7% at 6.45 microns (P Ͻ 0.007), and 52% at 7.7 microns (P Ͻ 0.02). The lateral in monolayers. Nine days after seeding, when cells matured into a ‘‘skin-like’’ structure, they were thermal damage also decreased as template thermal conductivity increased at a fixed wavelength irradiated using a UVB source (Ultralite bulbs) with a Kodacel filter. UVB (2–50 mJ per cm2) did (7.7 microns). Compared to the thermal damage produced without a template, we observed a not alter collagen synthesis in either culture system. TNF-01α (0.1–100 ng per ml) decreased 74% reduction using copper (P Ͻ 0.0001), a 52% reduction using aluminum (P Ͻ 0.02), a 9% collagen synthesis in a dose-dependent manner in monolayers, but not in gels. In contrast, TNF- reduction using glass (P Ͻ 0.52), and a 47% increase using plexiglass (P Ͻ 0.0001). The copper 01α decreased collagen type I mRNA expression in both monolayers and collagen gels as shown and aluminum heat-conducting templates therefore provided a significant decrease in lateral by RT-PCR. Similar results were found for IL-101β (0.5–100 ng per ml). Therefore, it appears thermal damage which depended upon the laser wavelength and template composition. This novel that UVR may decrease collagen synthesis by fibroblasts indirectly via cytokines rather than by heat-conducting template holds promise for reducing tissue damage with infrared laser surgery. direct effects on the cells.

813 814 Actinic Prurigo: Clinical Features and HLA Associations in a Canadian Inuit Population The Antioxidant N-acetyl Cysteine Increases the Content of the Endogenous Antioxidant M. C. Wiseman, P. Orr, S. Macdonald, M. Schroeder, J. W. Toole Glutathione and Prevents UV-induction of Matrix Metalloproteinases in Human Skin In Vivo Section of Dermatology, University of Manitoba, Winnipeg, MB; Division of Dermatology, S. Kang, G. Fisher, E. Duell, Y. Wan, J. Varani and J. Voorhees University of British Columbia, Vancouver, British Columbia; and Department of Medicine, Department of Dermatology, University of Michigan, Ann Arbor, Michigan Community Health Sciences and Pediatrics and Child Health, University of Manitoba, Winni- Ultraviolet (UV) irradiation activates cell surface receptors and their associated signaling machinery peg, MB thereby upregulating AP-1 and its target, matrix metalloproteinase (MMP) genes in human skin. Actinic prurigo is an idiopathic familial photodermatosis of First Nation and Inuit populations. MMP-mediated dermal matrix degradation is a major contributor to premature skin aging The objectives of this study were to examine the clinical features and HLA associations in an (photoaging). Evidence suggests that responses of cells to UV are initiated by generation of reactive Inuit population. Thirty-seven Inuit subjects with actinic prurigo from the Keewatin region of oxygen species (ROS) and agents that neutralize ROS (antioxidants) can inhibit UV responses. the North-west Territories were identified, subjected to a questionnaire, and underwent a complete We investigated the ability of an antioxidant N-acetyl cysteine (NAC), which can be converted cutaneous examination. Other causes of photosensitivity were excluded on the basis of history, into the endogenous antioxidant glutathione, to affect UV-induction of MMP in human skin and autoantibody and porphyrin screening. To minimize the possibility of latent photosensitivity, in vivo using a UVB/A2 source (filtered to remove UVC). In vehicle treated skin, both the inclusion criteria for the 43 HLA controls included age greater than 40 and the absence of a reduced (GSH; 84%) and the oxidized (GSSG; 16%) forms of glutathione were detected. 20% family history of photosensitivity. HLA – ABC (Class I) and DRB (Class II) low resolution typing NAC applied under occlusion for 24 h increased the GSH level by 50% (P Ͻ 0.05) and eliminated was performed using MICRO SSP Generic HLA Class I DNA typing and Amplicor for Class II the oxidized form (GSSG) completely (P Ͻ 0.05; n ϭ 6). Compared to vehicle, NAC prevented typing. Subjects were 81.1% female and 67.6% had a family history of photosensitivity. The UV-induction of collagenase and 92 kDa gelatinase by 63% and 46%, respectively (P Ͻ 0.05; n ϭ average age of onset of photosensitivity was 29 y and only 27% of subjects had an overall trend 6). Prior to UV, NAC treatment of at least 7 h was required to prevent MMP induction. NAC towards improvement in photosensitivity. Pruritus was present in 94.6% of subjects and 100% pretreatment did not block UV-autophosphorylation of epidermal growth factor-receptor (western experienced a seasonal variation. The rash began between February and May, and resolved between blot; n ϭ 6), suggesting that the site of GSH action is distal to receptor activation by UV. NAC July and November in 91.9% and 89.2%, respectively. Eye involvement was reported in 62.2% had no effect on UV-induced erythema. Thus, in human skin in vivo, prior application of the of subjects and 18.9% reported involvement of nonexposed skin. Physical examination revealed antioxidant NAC, which increases the content of endogenous antioxidant GSH, prevents UV- involvement of the face (64.9%), lip (32.4%), dorsal aspect of the hand (24.3%), ear (13.5%), neck induction of MMPs. These data suggest that antioxidants such as NAC may prevent photoaging. (13.5%), and extensor aspect of the arm (10.8%). Statistically significant HLA associations were not identified. There was a suggestion that HLA B*07 was a protective antigen (2.7% subjects, 14% controls, P ϭ 0.078). This study has better characterized clinical features of actinic prurigo in the Inuit and has demonstrated that it is a pruritic disease of adulthood that tends to worsen over time, and resolve in winter months. Both photo-exposed and nonexposed skin may be affected. HLA associations identified by past studies in First Nation populations, were not found in this Inuit population. A trend for a HLA antigen (B*07) that is protective against actinic prurigo is a novel finding

815 816 Effect of Ultraviolet Radiation on Expression of p53 and Induction of Apoptosis In Vivo in Solar Damage Identified by Ultrasound in Skin Cancer Patients and Controls Human Skin P. Lenane, M. Mabruk,* M. Murphy,* P. M. Cann, M. Leader,* E. Kay* and G. M. Murphy M. Murphy,* M. Mabruk,* P. Lenane, P. McCann, P. Buckley, M. Leader, E. Kay and G. Murphy Department of Dermatology and *Pathology, Beaumont Hospital, Dublin, Ireland Departments of Dermatology and Pathology, Beaumont Hospital, Beaumont, Dublin, Ireland High resolution ultrasound is a noninvasive method of assessing changes in human skin. In an Ultraviolet radiation (UV) is a complete carcinogen. UV is also a potent inducer of apoptosis. effort to identify the amount of solar induced damage present in patients with different types of The molecular events associated with UV induced apoptosis are slowly being unravelled in vitro. skin cancer we undertook ultrasound measurements in patients with three tumour types.52 patients p53 protein is however, a critical component of the cell regulatory mechanism. The aim of these were studied on three sun exposed and one protected site including the right forearm, right studies was to determine the time course and dose response for p53 expression and apoptosis in temple, the glabellar region of the forehead and unexposed buttock skin. This group included 36 response to solar simulated radiation in vivo in human skin. Five volunteers were recruited for this patients with basal cell carcinomas, seven with squamous cell carcinomas, three with melanomas study. The minimal erythema dose (MED) was determined for each volunteer using graded doses and seven control patients. Age ranged from 28 to 81 y. We identified a subepidermal low of UV from an Oriel solar stimulator on sun-protected buttock skin. Adjacent areas 4 cm2 were echogenic band on the forearm of 12 patients. Three of whom also had one on the forehead. exposed to 0.5, 1, 2, 3 MEDs. Biopsy specimens (4 mm) were taken from these sites at 4.5 These 12 patients included one female with melanoma, a second with squamous cell carcinoma, (volunteer A), 9 (volunteer B), 24 (volunteer C), 33 (volunteer E), 48 (volunteer F) h after UV one male and six females with basal cell carcinomas and three control patients. All 12 patients had irradiation and from an adjacent control nonirradiated site in each case. Induction of apoptosis at photodamage clinically. The proposed hallmark for solar elastosis on ultrasound is the presence of a single cell level was determined histologically using Haemotoxylin & Eosin staining and the In a subepidermal low echogenic band. It is felt to be secondary to the accumulation of glycosaminogly- Situ Cell Death Detection Kit (Boehringer Mannheim). p53 positive cells were detected cans and water in association with the elastotic degeneration of collagen and the accumulation of immunohistochemically on formalin fixed skin biopsies using DO-7 monoclonal antibody for abnormal elastic tissue. In our study sun shielded areas were consistently normal. Striking ultrasound p53. Increased numbers of apoptotic cells and p53 positive cells in volunteers exposed to 3 MED change including a subepidermal low echogenic band which correlated best with age and sun were seen in comparison to lower doses. The maximum number of p53 positive cells was seen at exposure rather than tumour type. High resolution ultrasound examination of the skin may 9–24 h whereas highest number of apoptotic cells occurred at 33 h post UV irradiation. These provide one noninvasive method of assessment of severe photodamage in patients and may prove data indicate that expression of p53 precedes induction of apoptosis in vivo in human skin and useful in epidemiological studies. both effects are dose dependent. (*Joint first author.) VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 659

817 818 Ultraviolet Irradiation Increases Matrix Metalloproteinase-8 Protein in Human Skin In Vivo EGFR-Dependent Bcl-xL Expression Protects Keratinocytes Against UVB-Induced Cell Death H. Choi, Z. Bata-Csorgo, Z. Wang, S. Kang, G. Fisher and J. Voorhees M. Jost, F. Gasparro and U. Rodeck Department of Dermatology, University of Michigan, Ann Arbor, Michigan Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Philadelphia, Photoaging results from Ultraviolet (UV) induced degeneration of collagen in the dermal Pennsylvania extracellular matrix via induction of various matrix metalloproteinases (e.g. MMP-1) in skin cells. Regulation of keratinocyte survival is crucial to skin homeostasis. Multiple cell surface receptors In this study, we examined whether UV irradiation also results in increased neutrophil collagenase have been implicated in this process. Stimulation of these receptors transmit either pro-apoptotic (MMP-8) expression in human skin in vivo. Skin exposed to UV (2MED, UVB source filtered to (Fas/CD95, TNF-R) or antiapoptotic (growth factor receptors) signals that regulate activation of remove UVC) were analyzed for MMP-8 mRNA by RT-PCR, and protein by immunohistology, the intracellular apoptosis machinery. Recently we have shown that the epidermal growth factor ELISA and Western analysis. We also investigated the effect of RA (0.1%), and temovate (0.05%) receptor (EGFR) transmits survival signals for cultured keratinocytes. Blockade of the EGFR with on MMP-8 protein expression after UV irradiation. ELISA revealed that MMP-8 protein was an antagonistic antibody (mAb 425) enhanced the susceptibility of keratinocytes to undergo increased several folds 8 and 16 h post UV. MMP-8 protein levels returned to basal level 48 h apoptosis upon extracellular stress, e.g. detachment from substratum, by downregulating expression post UV (n ϭ 5). Similarly by western analysis, MMP-8 protein was increased 9-fold and 8.7- of the antiapoptotic Bcl-2-homologue, Bcl-xL. Here, we demonstrate that EGFR-engagement fold 8 and 16 h post UV, respectively (n ϭ 4). Immunohistology analysis showed that MMP-8 does not affect expression of pro-apoptotic members of the Bcl-2 family (i.e. Bad, Bak, and Bax). was expressed by neutrophils in the papillary dermis 4 and 8 h post UV with appearance of such In addition, we provide evidence that EGFR activation protects keratinocytes from UVB-induced cells in the epidermis 24 h post UV. Temovate partially prevented the UV-induced appearance of cell death. This effect is due, in part, to upregulated Bcl-xL expression since immortalized MMP-8 protein and the infiltration of neutrophils. However, RA did not inhibit MMP-8 keratinocytes (HaCaT) transfected with Bcl-xL are protected against UVB-induced apoptosis. appearance. Based upon RT-PCR, there was no increase in MMP-8 mRNA expression after UV These results indicate that, in human keratinocytes, activation of Fas/CD95 and TNF-R by UVB exposure. In conclusion, in response to a single dose of 2 MED UV, MMP-8 protein was increased exposure is associated with a type II apoptotic pathway (Scaffidi et al. EMBO J 1998, 17:1675–1687). after 8 and 16 h and this increase was associated with the appearance of neutrophils in the UV- exposed skin which was suppressed by temovate but not RA pretreatment. The lack of induction of MMP-8 mRNA suggests that the increased MMP-8 protein levels are due to preformed MMP- 8 in infiltrating neutrophils.

819 820 Involvement of Epidermal Growth Factor Receptor (EGFR)-Tyrosine Kinase Pathway in Regulation of Cyclin Dependent Kinase CDK1, Cyclin A, Cyclin B, Topoisomerase II01α, and Photodynamic Therapy-Mediated Cell Death RAD51 by Photodynamic Therapy in Human Epidermoid Carcinoma Cells A431 K. Kalka, N. Ahmad, T. Kermode and H. Mukhtar N. Ahmad, M. S. K. Kalka and H. Mukhtar Department of Dermatology, Case Western Reserve University, Cleveland, Ohio Department of Dermatology, Case Western Reserve University, Cleveland, Ohio Photodynamic therapy (PDT), a relatively new therapeutic procedure for the management of Photodynamic therapy (PDT) is a promising investigational treatment modality for a variety of cutaneous cancers and other skin disorders, involves the administration of a photosensitizing cutaneous neoplasms and other skin disorders. We have earlier shown that silicon phthalocyanine compound followed by illumination of the target lesion with visible light. We have earlier shown (Pc4)-PDT results in WAF1/p21-mediated G0/G1-phase arrest followed by apoptosis in human that silicon phthalocyanine (Pc4)-PDT of human epidermoid carcinoma cells A431 results in a epidermoid carcinoma cells A431 (Proc Natl Acad Sci 95:6977–82, 1998). More recently, we cell growth inhibition, G0/G1-phase cell cycle arrest and apoptosis. In the present study we demonstrated the involvement of pRb/E2F-DP machinery during Pc4-PDT-mediated cell cycle investigated the involvement EGFR-tyrosine kinase pathway during the antiproliferative effects deregulation in A431 cells (Oncogene, in press). Here we extended our studies to investigate the of Pc4-PDT in A431 cells. EGFR has been implicated in many human tumors including squamous involvement of other cell cycle regulated proteins involved in cellular proliferation, DNA synthesis/ cell carcinoma of the skin. The inhibition of EGFR has also been shown to induce tumor cell repair, DNA topology, during PDT-mediated apoptosis. Western blot analysis revealed significant destruction via apoptosis, which makes it an attractive target for anticancer therapy. Western blot down-regulation of the cell cycle regulatory proteins cyclin A and B1, and cyclin dependent analysis demonstrated a significant inhibition of EGF-mediated increase in EGFR following Pc4- kinase CDK1 at 3, 6, and 12 h post-PDT. Interestingly, these proteins are largely involved in the PDT at 0.25, 0.5, 1, 3 and 6 h. EGFR overexpression is shown to be accompanied by increased cell cycle regulation at S-and G2-M phases. Pc4-PDT also resulted in inhibition of CDK1- phosphorylation of the tyrosine kinase domain of EGFR, resulting in continuous cell signaling, associated kinase activity. Western blot analysis also revealed that Pc4-PDT, at similar times post- uncontrolled cell proliferation and cancer growth. We determined the effect of Pc4-PDT on PDT, results in an inhibition of RAD51, which is a recently identified protein that is proposed tyrosine phosphorylation of EGFR. Immunoprecipitation of EGFR followed by immunoblotting to play a key role in the repair of DNA-double strand break. We also studied the modulation of with antiphosphotyrosine antibody revealed that Pc4-PDT also results in a significant decrease in topoisomerase II01α by Pc4-PDT because DNA topoisomerase II, regarded to be essential for EGF-mediated phosphorylation of EGFR at tyrosine. Furthermore, Pc4-PDT also resulted in an survival of all eukaryotic cells, is a nuclear protein known to modulate DNA topology during inhibition of EGF-mediated increase in the protein expression of Shc, an immediate downstream several metabolic processes and is shown to be a target of several antitumor drugs. Pc4-PDT molecule in EGFR-tyrosine kinase pathway. Taken together, these data suggest the involvement resulted in a significant inhibiton of topoisomerase II01α at 3, 6, and 12 h post-PDT. 01[captau]aken of EGFR-tyrosine kinase pathway during PDT-mediated cell growth inhibition, cell cycle together, these data suggest that Pc4-PDT-caused cell growth inhibitory effects are mediated via deregulation and apoptosis. It is likely that EGFR-inhibitors could be useful in enhancing the multiple pathways responsible for cell cycle regulation, DNA synthesis/repair, DNA topology, efficacy of PDT of skin cancers. ultimately leading to apoptosis.

821 822 Modulation of UVB-Stimulated Tumor Necrosis Factor but not Cyclooxygenase II Production Activation of the Epidermal Growth Factor Receptor is Required for Ultraviolet B-Induced by the Epidermal Platelet-activating Factor Receptor Apoptosis in Human Keratinocytes L. Dy, Y. Pei and J. Travers S. Hurwitz and D. Spandau; Departments of Dermatology, Pharmacology and the Herman B Wells Center for Pediatric Departments of Dermatology and Biochemistry, Indiana University School of Medicine, Indiana- Research, Indiana University School of Medicine, Indianapolis, Indiana polis, Indiana Increasing evidence suggests that the phospholipid platelet-activating factor (1-alkyl-2-acetyl- The reaction of human keratinocytes to ultraviolet B (UVB) radiation is determined by the glycerophosphocholine; PAF) is involved in keratinocyte biology. Our previous studies have integration of survival and apoptotic signals. Our laboratory is interested in defining the parameters demonstrated that human keratinocytes both synthesize PAF and express a functional PAF receptor that control the fate of keratinocytes irradiated with UVB. Previous data that was generated in (PAF-R) linked to the biosynthesis of numerous cytokines, including interleukins IL-6, IL-8 and other labs have shown that the epidermal growth factor (EGF) receptor can be activated by UVB eicosanoids. To study the role of the PAF-R in human keratinocyte function, we have stably in the absence of EGF. UVB-dependent activation of the EGF receptor results in the subsequent transduced the human PAF-R-negative epidermal cell line KB with the human wild-type PAF- activation of JNK and AP-1. We asked what effect the inhibition of UVB-dependent activation R using a recombinant replication-defective retrovirus. Because UVB can stimulate PAF biosynthesis of the EGF receptor would have on UVB-induced apoptosis. Primary human keratinocytes were in epidermal cells, we used this novel model system to assess whether the epidermal PAF-R is treated with genistein, a nonspecific inhibitor of tyrosine kinase activity, or 4,5-dianilinophthalimide involved in UVB-induced tumor necrosis factor alpha (TNF) production. Treatment of PAF-R- (DAPH), a selective inhibitor of the EGF receptor tyrosine kinase, 30 min prior to UVB expressing KB cells (but not control KB cells transduced with blank vector) with a PAF agonist irradiation. Both genistein and DAPH inhibited UVB-induced apoptosis. As expected, DAPH resulted in increased TNF mRNA and protein. Treatment of KB cells with UVB at doses which also inhibited the UVB-dependent activation of the EGF receptor; therefore, the UVB-dependent can generate PAF biosynthesis (200–400 J per m2) resulted in an exaggerated TNF mRNA activation of the EGF receptor is required for the apoptotic signal. We also demonstrated that accumulation and protein release in the PAF-R-expressing over control KB cells. However, UVB UVB radiation induced a persistent activation of the EGF receptor following a single UVB irradiation upregulated cyclooxygenase II mRNA by similar amounts in both PAF-R-expressing exposure. Downstream effectors regulated by this activation of the EGF receptor include the p38 and control KB cells. These findings suggest that the PAF system is a physiological target for MAP kinase. Inhibition of p38 MAP kinase activity by SB203580 also prevented UVB-induced UVB, and could be involved in UVB-induced keratinocyte cytokine production. apoptosis in human keratinocytes. We propose that the UVB-dependent activation of the EGF receptor and the subsequent activation of p38 MAP kinase are required for UVB-induced apoptosis in human keratinocytes. 660 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

823 824 Impaired Ultraviolet B Induced Cytokine Induction in Xeroderma Pigmentosum Fibroblasts Porphyria Cutanea Tarda and Hepatitis C Virus: A Case-Control Study and Meta-Analysis of H. Suzuki, G. Shivji, B. Wang, K. Kraemer* and D. Sauder the Literature Division of Dermatology, Sunnybrook Health Science Centre, University of Toronto, Toronto, T-Y. Chuang, R. Brashear and C. Lewis Canada. *Laboratory of Molecular Carcinogenesis, National Cancer Institute, Bethesda, Maryland Department of Dermatology, Indiana University School of Medicine, Indianapolis, Indiana Xeroderma pigmentosum (XP) is a rare, autosomal recessive disease in which patients develop Porphyria cutanea tarda (PCT) has been reported in patients infected with hepatitis C virus (HCV) excessive solar damage at an early age and have a 1000-fold increased risk of developing cutaneous since early 1990. However, many such case reports were confounded by selection bias and a high neoplasms. These neoplasms are usually thought to be related to defective repair of UV damage prevalence of HCV in populations studied. Further, only two small case series were reported from to DNA with resulting somatic mutations. We are examining the possible role of immune the U.S. We designed a case-control study to control such bias and confirm that the association deficiency in UV carcinogenesis in XP. Recently, several studies have shown impaired immune between PCT and HCV was common in the U.S. function in XP patients including reduced natural killer cell activity and enhanced immunosuppres- We reviewed the medical records of Wishard Memorial Hospital, a county hospital serving sion by UV. XP can be classified into seven complementation groups (A-G) with defects in metropolitan Indianapolis, Indiana, to perform a case-control study of 26 patients with PCT (as different DNA repair genes. We found that in XPA, XPD demonstrated increased apoptosis case) against 51 patients on methotrexate for psoriasis (as control-1) and 149 756 regional volunteer (18.2%, 17.8%, respectively) compared to normal human fibroblasts (NHFB) (6.1%) 24 h after 50 blood donors (as control-2). All 77 consecutive patients with PCT and Psoriasis were seen and mJ per cm2 UVB irradiation. In order to clarify the role of cytokines in XP, we examined post- treated at Wishard Memorial Hospital during the period from 1987 to 1997. Regional blood UVB cytokine mRNA expression in six XP fibroblasts strains in complementation groups XPA, donor data was obtained from the Central Indiana Regional Blood Center. Main outcome XPC and XPD. We measured mRNA by use of quantitative RT-PCR. We found that 24 h after measures were the HCV antibody titers and other liver abnormalities. We then performed a 50 mJ per cm2 UVB mRNA expression of IFNa was 15%–70% of that of NHFB and the weighted meta-analysis of 17 reports from the literature which had at least 17 patients in their fibroblasts from unusual XPC patient showed the lowest level. IFNb and IL6 expression was study populations. 10%–50% and 25%–50% of NHFB, respectively, and XPA cells showed the lowest level. These Sixteen (94%) of 16 PCT patients tested for HCV were antibody positive. Of five patients tested results demonstrate a deficient response of XP fibroblasts to UVB in terms of cytokine induction. for HCV in the psoriasis group, none were HCV antibody positive (P ϭ 0.0002, two-sided, This immune deficiency may play a role in the high frequency of skin cancer in XP patients. Fischer’s exact test). Of 149 756 regional volunteer blood donors in central Indiana in 1997, 255 (0.17%) were HCV antibody positive (P Ͻ 10–5, two-sided Chi-square test). For geographical comparison, meta-analysis of the literature demonstrated regional prevalence of HCV in PCT patients as follows: Northern Europe, 17%, Australia/New Zealand, 20%, and Southern Europe, 65%. We concluded that although a marked geographical variation was found in the prevalence of HCV in PCT patients, a large percentage of U.S. patients with PCT did have hepatitis C infection. Our results emphasize the need for clinicians to actively look for HCV in patients with PCT.

825 826 Occupational Hand Dermatitis and Skin Protection Habits during Vocational Training in Health Racial Differences in Skin Irritation Responses Between Caucasian and Asian Populations Care Workers and Hairdressers M. K. Robinson A. Bauer, A. Seidel, R. Bartsch, R. Schiele and P. Elsner The Procter & Gamble Co., Cincinnati, Ohio Department of Dermatology, Department of Occupational, Social and Environmental Medicine, One of the more confounding issues in dermatology relates to racial differences in susceptibility Friedrich-Schiller-University, Jena, Germany of human subjects to skin reactions resulting from chemical or physical trauma. There is some Starting in August 1992, 169 hairdressers and 255 nursing apprentices were included in a historical evidence to support a greater resistance of Blacks versus Caucasians to skin irritation or prospective follow up study in East Thuringia, Germany. The apprentices were interviewed and skin allergy, likely due to a more substantial barrier to chemical penetration. A heightened examined in a standardized way at the beginning of the training (hairdressers and nurses), after 6 sensitivity among Asian subjects (versus Caucasians) has been suggested; however, the evidence mo (hairdressers), after 1 y (nurses) and at the end of their training (hairdressers and nurses) after published to date is not very compelling. Part of the difficulty has been the lack of concurrent 3y. studies. The focus of this investigation was to directly compare skin irritation responses between Preoccuppational hand dermatitis was reported by 6.7% (n ϭ 17) of the nursing and in 1.8% (n ϭ Caucasian subjects and different subpopulations of Asia subjects (Chinese, Japanese) to determine 3) of the hairdressing apprentices. At the initial examination 15.3% (n ϭ 34) of the nurses and if significant differences in response patterns would emerge. Three separate studies were conducted. 17.8% (n ϭ 30) of the hairdressers showed hand dermatitis. At the first follow up frequency of Most of the work involved elicitation of mild skin irritation responses to relative high concentrations hand dermatitis in both professions rose to 24.9% (n ϭ 49) in nurses and 39.5(n ϭ 60) in of test chemicals using a novel acute irritation patch test protocol for chemical irritation hazard hairdressers. A minor reduction of the frequency of hand dermatitis was found after3yinboth identification. Using this protocol, Caucasian and Chinese subjects were exposed under patch for professions. Simultaneously skin protection habits changed. Glove wearing and use of skin up to 4 h to 20% sodium dodecyl sulfate (SDS), 10% acetic acid (HAc), 0.5% sodium hydroxide, protection creams and skin care products increased. After one year of training 57% of the 100% decanol, or water. No significant differences were seen in the cumulative incidence of hairdressers and 73.3% of the nurses used gloves regularily. positive responders to any of the test chemicals across the two populations. A second study of Nevertheless skin damage is common in occupations with skin hazards like nursing and hairdressing. identical design, conducted with subjects prescreened and divided into subpopulations based on There is still a tremendous demand for research to analyse the work profiles and develop adapted sensory reactivity to 10% lactic acid, produced similar results. The third study compared Caucasian skin care procedures in future. and Japanese subjects for responses to 20% SDS, 10% HAc, 100% octanoic acid, 100% decanol, and water. In addition, a concurrent test was conducted comparing 14 d cumulative irritation responses to 0.025% to 0.3% SDS. In both the acute and cumulative irritation tests, the Japanese subjects showed a tendency to respond faster than the Caucasian subjects. Significant differences in response were seen at the early time points in the acute irritation test, and with the lowest concentration of SDS (0.025%) in the cumulative irritation test. These results may point to subtle differences in population reactivity. However, the results need confirmation and extension to determine if they represent true population differences or simply reflect the inherent variation in response across human subjects.

827 828 Validation of the Dermdex: the First Quality-of-Life Instrument for HIV(ϩ) Patients with Use of Unconventional Medicine by Dermatology Patients for Treatment of Inflammatory Skin Disease Skin Diseases K. Aftergut, R. Wilson, T. Carmody and P. Crug, Jr B. Miller, M. Kumar and H. Faust Department of Dermatology, University of Texas Southwestern Medical Center, Dallas, Texas Department of Dermatology, Indiana University, Indianapolis, Indiana Quality-of-life (QOL) instruments capture patients’ perspective of their health, which can differ Unconventional medicine (also known as alternative medicine) is defined as those medical systems, significantly from the view of their physicians. Few QOL surveys have been developed for patients professions, practices, interventions, or theories that are not currently part of the dominant medical with dermatologic problems in general. None have been targeted for HIV(ϩ) patients with skin system in the United States. This includes over 300 different categories of intervention such as disease, whose health assessment often requires particular questioning. To fill this gap, we created chiropractic, acupuncture, herbal remedies and other dietary modification. They often effect a 10-item QOL questionnaire, termed the DERMDEX. The questionnaire was validated through disease course and may impact on diagnosis and treatment of the disease. The use of unconventional cross-sectional and longitudinal surveys of HIV(ϩ) and HIV(–) individuals, in parallel comparison remedies by dermatology patients to treat inflammatory skin diseases has not previously been with the SKINDEX-29 (an established instrument for general skin conditions). DERMDEX was studied. A survey was designed to quantify the type and number of dermatology patients in an also administered to HIV(ϩ) patients with disseminated molluscum contagiosum (MC), seborrheic outpatient setting using unconventional therapies, to determine what treatments they are using dermatitis (SD), and eosinophilic folliculitis (EF). Outcome measures included reproducibility, and why they chose them, and to assess whether treating physicians are aware of their regimens. internal consistency reliability, capacity to discriminate among different disease states, and A total of 147 patients were surveyed of whom 44% stated they used a some remedy prior to responsiveness to therapeutic intervention. DERMDEX scores were reproducible over a 7-d being seen by a dermatologist. Although the goal of the survey was to document unconventional interval (Pcarson Coefficient) and were internally reliable (Cronbach a Coefficient). Results of medicine use, much of the data collected really reflected home remedy usage. Among the items these two measures were comparable to those of SKINDEX-29. Moreover, DERMDEX used, emollients, vitamins and herbal products, and rubbing alcohol were most commonly used discriminated between patients with MC or SD and those with EF, the latter group having by patients. Patients reported they conceived of the intervention by themselves most frequently. significantly worse QOL parameters (Kruskall-Wallis Test). Finally, DERMDEX detected significant A family or friend and the news media were the next most frequently reported sources of QOL improvement in patients with EF who were treated with itraconazole, but not with information for treatment advice. Fifty-five per cent of the patients did not reveal their use of ivermectin or metronidazole (Kruskall-Wallis Test), an outcome that ran parallel with results of self-treatments to physicians previously treating them. In summary, this study shows that a large therapeutic efficacy. We conclude that the DERMDEX is a valid and reliable instrument for proportion of dermatology outpatients use home remedies and that a significant proportion of the measuring QOL in HIV(ϩ) patients with skin disease therapies used reflect a belief in alternative medicine in this patient population. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 661

829 830 Epidemiology of Bullous Pemphigoid in Germany Clinical Re-Evaluation of Erythema Exsudativum Multiforme Majus and Stevens–Johnson B. Rzany, K. Partscht,† M. Jung, W. Kippes,* C. Prudlo,‡ D. Mecking,§ L. Bu¨chner‡,M. Syndrome Resulting in a Different Etiology Schuhmann, L. Bu¨dinger,ϩ M. Sticherling,** M. Hertl,ϩ H. Kaiser,§ M. Meurer,‡ D. Zillikens* M. Mockenhaupt, W. Schro¨der, J. Schlingmann, B. Schneck, O. Hering and E. Scho¨pf and G. Messer† Dokumentationszentrum schwerer Hautreaktionen, Department of Dermatology, University of Department of Dermatology, Universities of Mannheim, Aachen,ϩ Bonn,§ Dresden,‡ Kiel,** Freiburg. Germany Mu¨nchen,† Wu¨ rzburg* In 1990 a population-based registry for all hospitalized cases of toxic epidermal necrolysis (TEN), Large studies on the epidemiology of bullous pemphigoid are limited. None has ever been Stevens–Johnson syndrome (SJS) and erythema exsudativum multiforme majus (EEMM) was performed in Germany. In a multicenter effort a retrospective patient cohort was established of established in Germany. Whereas overlaps between SJS and TEN have been observed, a clear seven departments of dermatology to study these patients in detail. Patients were defined either distinction between EEMM and SJS, which has been proposed according to the original description by discharge diagnosis or by diagnosis of the immunodiagnostic laboratory. The diagnosis was of both entities, may be sometimes difficult. EEMM presents in a typical way with so-called validated based on direct and indirect immunofluorescence microscopy. Entry point in the cohort typical targets on the limbs, whereas atypical features with disseminated targets on other parts of was the first hospitalization. For data assessment an extensive standardized questionnaire was the body appear. In contrast, SJS is characterized by atypical targets and purpuric macules – developed and statistical analysis was done by SAS. So far a total of five centers has been analyzed. according to the original report – in a wide distribution, focussed on the trunk. According to the Age and gender distribution was similar between most centers. Alltogether 377 patients with BP type of lesions, an atypical distribution, e.g. on the extremities, represents an atypical form of SJS. were recruited. 92% of the patients could be followed up. The average follow up (Median Approximately 700 cases of EEMM and SJS that have been ascertained by the registry and been [25%,75% quantile]) was 2.1 [0.5, 4.4] years. Forty-five per cent of the patients died, 26% within reviewed by an independent expert committee were reevaluated and categorized as typical or the first year. The mean age at admission (Ϯ standard deviation) was 76.0 Ϯ 11.8 years. The atypical forms according to the clinical features without information on possible risk factors. The mean age at death (Ϯ standard deviation) was 82.4 Ϯ 8.3 y. Fifty-four per cent of the patients involvement of mucous membranes was not used as a criterion, because in the major type of were female, 23% presented with oral involvement. Twenty per cent of the patients reported a EEM as well as in SJS hemorrhagic erosive lesions of the mucosa are observed. The reevaluation malignancy. In 22% of the patients a type II diabetes was recorded at admission. Other autoimmune process separated 162 typical cases of EEMM from 120 atypical. In comparison, 278 typical and diseases (mostly rheumatoid arthritis and multiple sclerosis) were recorded in 5%. 82 atypical cases of SJS were seen. While patients Ͻ20 y predominate in atypical EEMM (54%) These results confirm that BP is a disease of the aged. A large number of BP-patients die in the and patients between 20 and 40 y in typical EEMM (48%), more than 60% of the patients with first year after diagnosis. Furthermore these descriptive data indicate an association of BP SJS were Ͼ40-y-old. As an etiologic factor for typical EEMM herpes simplex could be determined with malignancies. in 53%; in atypical EEMM the proportion of infections including mycoplasma, upper respiratory infection and flu was 67%. In contrast, 01µ50% of SJS-cases could be attributed to drugs previously known to induce severe skin reactions (carbamazepine, phenytoin, cotrimoxazole, allopurinol and piroxicam), whereas only 10% of both, typical and atypical EEMM-patients had been exposed to one of these drugs 2 wk prior to the onset of the reaction. The clinical pattern of severe skin reactions in the spectrum of erythema multiforme may provide information on the possible etiology of the reaction, allowing to decide on further investigations to clarify the etiologic cause.

831 832 Effect of Treatment of Helicobacter Pylori on Acne Rosacea Measurement Characteristics of a Condition-Targeted Questionnaire to Assess Health-Related J. Bamford, R. Tilden, J. Blankush and D. Gangeness Quality of Life in Women with Androgenetic Alopecia Dermatology Section and Division of Education and Research, SMDC Health System, Duluth, K. Dolte and C. J. Girman Minnesota Merck Research Laboratories, Blue Bell, Pennsylvania; Susan Hartmaier, Hartmaier Consulting, To evaluate the impact of recommended treatment for Helicobacter pylori on intensity of Dallas, Texas; Janet Roberts, NW Cutaneous Research Specialists, Portland, Oregon; Wilma symptoms among patients with Acne Rosacea. Randomized, double-blind, placebo-controlled Bergfeld, Cleveland Clinic Foundation, Cleveland, Ohio; Joanne Waldstreicher, Merck Research clinical trial carried out at the dermatology section of a large multispecialty clinic in the north Laboratories, Rahway, New Jersey central midwestern United Men and women aged 25 through 65 with Acne Rosacea and have A condition-targeted instrument to assess the quality of life in women with androgenetic alopecia tested positively with the urea breath test for Helicobacter pylori. Treatment of Helicopter pylori (AGA) was recently developed. Item generation, item reduction and pilot testing yielded a 25- (H.P.) infection with 14-day therapy using clarithromycin 500 mg orally, three times a day and item questionnaire that addresses aspects of hair loss that women with AGA have identified as omeprazole 40-mg orally once a day. Extent and intensity of Acne Rosacea, Quality of Life as relevant and important. Inclusion of the questionnaire in a clinical trial allowed the assessment of measured by a dermatological specific form, and overall health status as measured by the SF36 measurement characteristics of the questionnaire. form. 320 people presented to the Dermatology section with Acne Rosacea. Fifty of these people The Women’s Androgenetic Alopecia Quality of Life questionnaire (WAA-QoL) was completed were positive using the Urea breath test, and 44 agreed to participate in the study. While by 137 women in a year-long randomized, double-blind, placebo-controlled trial aimed at assessing improvement in the acne Rosacea was seen in almost all patients, there was almost no difference the effect of finasteride 1 mg per d in postmenopausal women with AGA (median age, 53; range, between the patients treated for H.P. and for those in the control cohort. The patients seemed to 41–60). Patients completed the WAA-QoL duringa2wkplacebo run-in period (week –2), at feel that their quality of life improved, but there was no difference between the treatment and baseline, and again 3, 6, 9, and 12 mo later. Factor analysis was used to explore domain structure. control arm into the magnitude of improvement. Health Status as assessed by the SF 36 varied Internal consistency was assessed at baseline using Cronbach’s alpha. Test-retest reliability was between the baseline and follow-up, with slight improvements and slight deteriorations being assessed at weeks –2 and baseline with the assumption that minimal change would occur during reported for all cohorts. There was improvement in Acne Rosacea for most of the patients in this this interval. Reliability was measured using the intraclass correlation coefficient (ICC) while study whether they were enrolled in the treatment or control cohort. It therefore appears unlikely agreement on individual items was assessed using weighted kappa statistics with quadratic weights. that Helicobacter pylori is either an important risk factor for Acne Rosacea, nor does it appear Correlations between scores and hair counts assessed in a representative 1 cm2 area of hair loss in that treating Helicobacter alleviates symptoms of Acne Rosacea. the frontal/parietal region of the scalp were calculated using Spearman rank correlation coefficients. Responsiveness was assessed by comparing treatment groups on changes in scores and individual questions. Nine questions were eliminated due to redundancy, resulting in a 16-item questionnaire. Test- retest reliability was excellent (0.89) while agreement on questions was substantial (0.66–0.85). The questionnaire demonstrated internal consistency (0.98). Although several questions were highly correlated with one another, they were retained because they addressed specific issues women with AGA identified as important. Scores were modestly correlated with hair counts (0.1). As no significant difference between treatment and placebo groups in hair counts was shown in this trial in postmenopausal women, responsiveness of the WAA-QoL could not be assessed. The WAA-QoL exhibits good content validity, internal consistency and test-retest reliability and may be useful in assessing the impact of health-related quality of life on women with androgenetic alopecia or therapeutic effects in clinical trials.

833 834 Risk Factors of Non-Melanoma Skin Cancer in United States Veterans Patients A Pilot Study Prevalence of Thorough Skin Examination for Early Detection of Melanoma and Review of Literature M. A. Weinstock, R. A. Martin, P. M. Risica, M. Berwick, T. Lasater, W. Rakowski, M. G. T-Y. Chuang and R. Brashear Goldstein and C. E. Dube´ Department of Dermatology, Indiana University School of Medicine, Indianapolis, Indiana Dermatoepidemiology Unit, VA Medical Center; Department of Dermatology, Rhode Island There are an estimated one million new nonmelanoma skin cancer (NMSC) cases annually in the Hospital; Center for Primary Care and Prevention, Memorial Hospital of Rhode Island; Memorial United States alone. While other studies with varying foci have evaluated risk factors in different Sloan-Kettering Cancer Center; and Departments of Dermatology, Community Health, and subsets of the general populace, none have examined veterans as a group with potentially unique Psychiatry and Human Behavior and Centers for Alcohol & Addition Studies and Primary Care exposures and risks. We want to identify risk factors of NMSC in U.S. veterans patients. & Prevention, Brown University; Providence, Rhode Island and New York, New York We conducted an Investigation of risk factors for skin cancer through questionnaire and physical Melanoma is a major public health problem for which early detection may reduce mortality. Since examination on 145 veteran patients with skin cancer and 59 veteran patients without a history melanoma is generally asymptomatic, detection requires skin examination. We sought to evaluate of skin cancer. We found that parents’ ethnicity, actinic keratosis on face or other anatomic sites, the frequency with which the general public either thoroughly examine their own skin or have solar elastosis of the neck, facial telangiectasias, age of first sunburn, and residency in Indiana were it examined thoroughly by their partners or health care providers. We conducted a random digit risk factors significantly associated with the development of skin cancer. Other possible risk factors dial survey of 200 Rhode Islanders. Only 9% performed a thorough skin examination (TSE) at include smoking and radiation therapy. least once every few months when questioned in detail, although over half of the sample reported We concluded that several risk factors would significantly increase the chance of developing skin conducting skin self-examination ‘‘deliberately and systematically’’. Participants were more likely cancer among veterans. These included ethnic background and solar damage of the skin among to perform TSE if they were women and if their health care provider had asked them to examine others. A review of the literature confirms these risks in the general populace, but also further their skin. Most participants reported that their health care provider never or rarely looked at study is warranted to address risk factors like smoking and radiation, particularly in veterans their back or the back of their legs, areas in which melanoma is most likely to arise. The frequency populations. Identification of pertinent risk factors will help to identify high risk individuals, allow of reported skin self-examination depends critically on the detail with which the question is asked. detection of new skin cancer at its earliest stage, and develop a profile of favorable lifestyle TSE by self or a partner is uncommon, and health care providers do not routinely examine the characteristics that reduce NMSC risk. areas of the skin on which melanomas commonly arise. 662 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

835 836 Quality of Life is Significantly Improved in Cutaneous T Cell Lymphoma Patients Who Respond Comparative Study of Dermatologists’ Perceptions of Factors Affecting the Practice of Dermatology R to DAB389-IL2 (ONTAK ) Fusion Protein S. Lui, L. Schumm and K. Soltani M. Duvic, T. Kunkel, E. Olsen, A. Martin, F. Foss, Y. Kim, P. Heald, P. Bacca and J. N. M. Anderson University of Chicago, Chicago, Illinois Cancer Center, Houston, Texas, North-western University, Chicago, Illinois; Duke University There have been no recent studies on the attitudes of dermatologists, particularly regarding issues Medical School, Durham NC, Washington University, St. Louis, Missouri, Tufts-New England of managed care with respect to the practice and research of dermatology. The study concentrated Medical Center, Boston, Massachusettts, Stanford, Palo Alto, California, & Seragen Inc., Worches- on the metropolitan area of a large city (Chicago) due to th e high density of specialists. ter, Massachusetts Dermatologists in Chicago and cities within a 40 mile radius were surveyed. 178 questionnaires DAB389-IL2 (Denileukin diftitox) belongs to a new class of receptor-active cytotoxic fusion were randomly mailed and dermatologists were asked to confidentially fill out surveys of 53 proteins, composed of diptheria toxin and the receptor binding domain of IL-2. It targets T cells questions pertaining to various factors of satisfaction in Likkert scale format. There was also a bearing the alpha chain of the IL-2 receptor, CD25. To determine response rate in CTCL, a place for additional comments. 110 total eligible responses were received, for a response rate of pivitol trial was conducted in 71 refractory patients. Objective response rate of 30%, included 62%. Likkert scale responses were converted to numerical analogs of –2 (strongly disagree), –1 seven CRs. No significant differences in patient characteristics, response, or duration of response (somewhat disagree), 0 (neither agree or disagree), 1 (somewhat agree), and 2 (strongly agree). were seen between two dose arms: 9 or 18 µg per kg. A trend towards a higher dose effect was Dermatologists generally expressed a very high degree of satisfaction, with 90% of all subjects seen in advanced patients: 10% vs 38%, P ϭ 0.07. Measurements of symptoms erythroderma, indicating current satisfaction with their jobservation Residents and fellows particularly expressed pruritus, global assessment, and quality of life by Fact-G were secondary end-points. A majority a high degree of satisfaction and optimism, with mean scores of 1.71 (95% confidence interval of patients who received DAB-IL2 had significant improvements in their pruritus and global skin [Cl], 1.56–1.87) and 1.54 (95% Cl, 1.37–1.72), respectively. The number of years spent practicing scores compared to baseline values (P ϭ 0.05). Physician’s global severity score improved in most, dermatology was a predictor of current satisfaction and optimism, exhibiting a trend of slight regardless of whether they met all of the criteria for partial response. The Fact-G quality of life decline with increasing years in practice (P Ͻ 0.001). A regression of years spent in dermatological score, composed of five subscores of well-being, was significantly improved from baseline in the practice vs. change in optimism yielded the greatest trend in decline. 85% of dermatologists also 21 responders and was maintained in all 71 patients who were treated. DAB389-IL2 (ONTAK) is felt that there was a decrease in freedom of practice, although freedom was not a significant a new type of therapy for CTCL which improved the quality of life and did not result in life- predictor of satisfaction. Over 95% of dermatologists experienced an increase in paperwork. threatening immunosuppression, common with other modalities. Overall, 83% of dermatologists felt that managed care leads to decreased overall quality of care for the patient. Although 74% of dermatologists felt that the field is experiencing a decrease in financial compensation, it was not statistically significant (P Ͻ 0. 132). In general, dermatologists appear to be very satisfied with the specialty, despite acknowledging pejorative changes in financial compensation, freedom of practice, paperwork, and the belief that managed care leads to decreased overall quality in healthcare. While there is a slight tapering of satisfaction among older dermatologists, the satisfaction rate has remained extremely high. Based on these results, we predict that residency programs will remain very competitive among medical students.

837 838 Dermatologists are More Likely to Cure Basal Cell Carcinomas Using Surgical Excision than Determinants of the Use of Alternative Methods in Allergic Patients: Results of a Population- Nondermatologists: Results from a Pilot Study Based Pilot Study A. S. Katz, A. B. Fleischer Jr, S. R. Feldman, D. M. Reboussin and B. Leshin T. Scha¨fer, C. Cramer and J. Ring The Westwood Squibb Center for Dermatology Research and the Departments of Dermatology Department of Dermatology and Allergy, Technical University, Munich, Germany and Public Health Sciences, Wake Forest University School of Medicine, Winston-Salem, Patients with chronic diseases increasingly seek help in alternative methods (AMs). This population North Carolina based pilot study tried to determine the characteristics of allergic patients who have experience The basal cell carcinoma (BCC) is the most common cutaneous malignant neoplasm treated by with alternative methods. Subjects were recruited from a case control study on allergies including physicians. We have previously shown that clinician experience in treating this cancer varies 800 adults (25–75-y-old) with and 800 without allergic sensitization to common aeroallergens as widely by physician specialty. Our objective was to determine predictive factors for the histologic measured by RAST. According to further dermatological examination and standardized interviews cure of BCC by surgical excision. We performed a 66-mo retrospective study of all BCC excision cases with atopic diseases (atopic eczema, hay fever, asthma) who reported experience with AMs specimens (1557 excision specimens submitted by 46 physicians from eight specialties) submitted were identiﬁed. Information on previous conventional therapy, source of information, kind of to the Wake Forest University Baptist Medical Center. Histologic evidence of tumor presence in AM, provider, costs, duration, and overall assessment were obtained by questionnaire. We received surgical margins of specimens was our main outcome measure. Presence of tumor at the surgical information from 66 persons with atopic eczema (n ϭ 3), hay fever (n ϭ 41), asthma (n ϭ 2) or margin was found in 217 (14%) specimens. Of physicians submitting specimens, dermatologists more than one atopic disease (n ϭ 20). Most patients had experience with autologous blood performed a mean (Ϯ SD) of 133 Ϯ 259 excisions per physician while nondermatologists injection (n ϭ 35) followed by acupuncture (n ϭ 23), and bioresonance (n ϭ 16). Almost all had performed an mean of 9.5 Ϯ 22 excisions per physician. Incomplete excisions occurred in 7% of previously received conventional therapy (85.9%). The majority got information on AMs by specimens submitted by dermatologists, whereas nondermatologists demonstrated incomplete friends and family (53.1%) or the current medical practitioner (42.2%). The actual AM was excisions in 38% submitted specimens. Regression found that greater experience predicted greater performed by the general practitioner in most cases (41.3%) followed by nonmedical practitioners cure rate. Logistic regression found that age and tumor size did not predict the likelihood of (34.9%), and medical specialists (27%). 9.5% of the patients practiced the methods themselves. incomplete excision, whereas physician specialty, tumor site, and sex predicted incomplete Most methods were applied once a week (37.3%) or more frequently for the duration of up to excision. Dermatologists were signiﬁcantly more effective at completely excising BCCs than were one month in 24.1% or up to1yin93.1%. Half of the patients reported that they were able to nondermatologists discontinue conventional therapy when using AMs. The effect of AMs lasted up to one month in 13.6% and 45.5% still noticed an effect. Thirty-nine per cent returned to the previous therapy when they stopped using AMs. The mean costs for a single treatment were about 57 US$ and the total costs averaged 790 US$. Most patients received full (38.3%) or partial (26.7%) reimbursement from their insurance companies. The overall effect of the AMs was assessed by the patients as good and very good in 75.4%. 21.1% reported no effect and only 3.5% observed a negative effect. The following main study will also focus on motivation and psychological background in comparison to patients without experience in AM.

839 Nondermatologists See Few Cases of any Individual Skin Disease in their Clinical Practice A. Fleischer Jr, C-R. Herbert and S. Feldman Westwood-Squibb Center for Dermatology Research and the Department of Dermatology, Wake Forest University School of Medicine, Winston-Salem, North Carolina Dermatologists have special expertise in treating skin disease when compared to nondermatologists. This may be due in part to the differential ongoing exposure to skin disease management of different medical specialties The purpose of this study is to quantify the relative exposure to dermatologic problems in the practices of dermatologists and primary care providers. Data on the frequency of diagnoses made at outpatient visits to physicians and the duration of these visits for both dermatologic and nondermatologic disorders were obtained from the 1990–94 National Ambulatory Medical Care Survey. The most common dermatologic problem seen by internists, family physicians and pediatricians is the 35th, 17th and 15th most common diagnosis seen by these providers, respectively. Dermatologists spend 930 outpatient hours per year caring for patients with dermatologic conditions, compared with 21, 53, and 56 h per year for internists, family physicians, and pediatricians. Dermatologists have far greater experience treating patients with skin problems and have the background training and continuing exposure required to maintain dermatologic skills at the highest level. VOL. 112, NO. 4 APRIL 1999 ABSTRACTS 663

Idea abstracts

I01 I02 Topical Steroid Phobia among Dermatology Outpatients with Atopic Eczema Prevalence and Predictors of Skin Disease in the Women’s Interagency HIV Study (WIHS) C. R. Charman, A. Morris and H. C. Williams P. Nfirmirani, T. A. Maurer, N. A. Hessol, T. G. Berger, P. Ngulen, A. Gurtman, A. M. Levine, Department of Dermatology, Queen’s Medical Centre, Nottingham, UK S. Nficei, B Solomon, M. Young and R.M. Greenblatt The aim of this study was to investigate the prevalence of topical steroid phobia as a potential WIHS Collaborative Study Group, USA cause of poor compliance with treatment in atopic eczema. 185 dermatology outpatients (6 mo– Our objective was to determine the prevalence and predictors of skin disease in a cohort of 68 y, mean age 13 y) with a mean duration of eczema of 11 years (range 3 mo–67 y) completed women with and at risk for HIV infection. We analyzed baseline data from a multicenter a questionnaire regarding their concerns about topical steroid treatment and knowledge of the longitudinal cohort (WIHS). All women had a baseline skin exam (10/94–10/95) by a health care different strengths of preparations available. 72% of patients worried about using topical steroids. provider. Diagnoses made by clinical exam were recorded. 24% of patients admitted to having not used topical steroids prescribed by a doctor because of 2018 HIVϩ women and 557 HIV– women were included in the analysis. Among HIVϩ women worries about steroid treatment. Skin thinning was the side-effect most commonly mentioned by the median CD4 count was 327 and the median viral load was 22 k. 63% of women with HIV patients as a cause for concern (32% of patients). 25% of patients worried about nonspecific long- had abnormal skin exams compared to 44% of HIV–women (OR 2.1, CI 1.7–2.5). HIVϩ women term effects/damage, whereas systemic absorption and effects on growth and development were were more likely to have Ͼ2 skin diagnoses (OR 4.0, CI 2.46.6). Independent predictors of an a cause for concern in 9% of patients. Of the 76% of patients who had used hydrocortisone, 69% abnormal skin exam in the HIVϩ were: African American race (OR 1.4, CI 1.2–1.7), recent or graded the preparation as weak or moderate in potency, but 31% were either unsure of the past injection drug use (OR 2.3, CI 23–3.8; OR 1.5, CI 1.2–1.9), CD4 count less than 50 (OR potency or graded it as strong or very strong. 22% (41 of 185) of patients had used both 1.7, CI 1.2–2.3) and higher viral loads (100K-499,999 OR 1.7, CI 1.4–23/ Ͼ499,999 OR 2.2, hydrocortisone and dermovate but of these only 61% (25 of 41) graded dermovate as more potent CI 13–3.2). The HIVϩ women had a significantly higher prevalence of folliculitis and onycho- than hydrocortisone. Common sources of information regarding topical steroids were the primary mycosis (P Ͻ 0.05). The diagnoses of molluscurn, herpes zoster, Kaposi’s sarcoma, and seborrheic care physician (33%), magazines (17%), family (13%) and friends (13%). These results demonstrate dermatitis were exclusive to the HIVϩ group. There was no difference in the prevalence of warts, that topical steroid phobia is a potentially important cause of poor compliance with treatment in tinea pedis, herpes simplex, psoriasis, or xerosis. atopic eczema and emphasise the need for better patient education regarding the different strengths This is the first large controlled study of skin disease in HIV that also includes CD4 count and of preparations available and their low potential for side-effects when used appropriately. virologic data. We observed a higher prevalence of skin disease-specifically folliculitis and onychomychosis-in HIVϩ versus HIV– women; moreover HIVϩ women were more likely to have Ͼ2 skin diagnoses. The skin diagnoses of molluscum, herpes zoster, Kaposi’s sarcoma and seborrheic dermatitis were exclusive to the HIVϩ group – thus serving as a marker of HIV infection in this population. Besides a CD4 count Ͻ 50, other important predictors of skin disease in HIVϩ women were higher viral load (Ͼ100K), injection drug use, or African American race.

I03 I04 Effect of 12 Years of Beta-Carotene Supplementation on Melanoma Skin Cancer in the Physician’s Melanoma Signs and Symptoms in Older Populations Health Study P. J. Christos, S. A. Oliveria, M. Berwick, D.. Guerry IV, D. E. Elder, M. Synnestvedt, J. Fine, U. Frieling, D. A. Schamberg, T. S. Kupper and C. H. Hemickens R. L. Barnhill and A. C. Halpern Division of Preventive Medicine and Dermatology, Harvard Skin Disease Research Center, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York; Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massa- Department of Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New chusetts York, New York; Yale University Cancer Center, New Haven, Conneticut; Johns Hopkins As the incidence of melanoma skin cancer (MSC) continues to increase, the search for possible University School of Medicine, Baltimore, Maryland; and Pigmented Lesion Group, University preventive modalities has intensified. Epidemiologic data on the potential role of beta-carotene in of Pennsylvania, Philadelphia, Pennsylvania the prevention of MSC are inconsistent, and there are as yet no data from randomized trials. The As with many other types of cancer, the incidence of melanoma increases with age. The impact Physicians Health Study (PHS) was a randomized, double-blind, placebo-controlled, 2 01ϫ 2 of advancing age may influence the reporting of specific signs and symptoms of melanoma. To factorial trial of beta-carotene (50 mg on alternate days) and aspirin (325 mg on alternate days) in assess the relationship of increasing age with the reporting of signs and symptoms of melanoma, the primary prevention of cancer and cardiovascular disease among 22,071 apparently healthy, we conducted a descriptive study. The study population was comprised of two groups: (i) 694 male, US physicians aged 40–84 y at baseline. During an average of 12 y of treatment and follow- stage I incident cases of melanoma diagnosed during September, 1972 to August, 1980 from the up of this population homogenous for high educational levels, there were 238 confirmed cases of Pigmented Lesion Group (PLG), University of Pennsylvania, and (ii) 624 stage I incident cases of MSC including melanoma in situ, 108 among the 11,033 randomized to beta-carotene and 130 melanoma diagnosed during January, 1987 to May, 1989 from the Connecticut Tumor Registry among the 11,032 randomized to placebo. Controlling for age and randomized aspirin assignment, (CTR). Information on clinical and pathologic variables for both study groups was obtained there was a nonsignificant 17% reduction of MSC in the beta-carotene group (relative risk [RR] including the following reported signs/symptoms of melanoma: bleeding, ulceration, itching, 0.83, 95% confidence interval [Cl] 0.64–1.07). Exclusion of the first 2 y after randomization did change in size, color, or elevation, and lesion tenderness. Logistic regression modelling was used not substantially change these results. There was also no significant evidence for benefit or harm to evaluate the relationship of older age (50 y) with the reporting of melanoma signs and symptoms. of beta-carotene on risk of MSC after stratification for use of vitamin supplements, body mass Analyses were adjusted for gender, lesion location, and tumor thickness. Older patients (50 y of index, or latitude. In subgroup data there was a reduced risk of MSC for past but not current age) were less likely (P Ͻ 0.05) to report early warning signs of melanoma including itching and smokers. Overall, the results of this large-scale randomized trial show no clear evidence for a change in elevation for both study populations (PLG, adjusted OR ϭ 0.48 & 0.60; CTR, adjusted chemoprotective effect of over 12 y of beta-carotene supplementation on the incidence of MSC. OR ϭ 0.58 & 0.47, respectively). Change in color of the lesion was also less likely to be reported among older patients, although it was not statistically significant (PLG, adjusted OR ϭ 0.82; CTR, adjusted OR ϭ 0.75). Ulceration of the lesion, a melanoma symptom characteristic of more advanced disease, was reported significantly more (P Ͻ 0.05) among older patients for both study populations (PLG, adjusted OR ϭ 2.20; CTR, adjusted OR ϭ 1.89). Bleeding of the lesion was also significantly elevated among older patients (adjusted OR ϭ 1.53), but only for patients attending the PLG. Our data suggest that older individuals are less likely to recognize early warning signs of melanoma, but more likely to report symptoms associated with advanced disease and poor prognosis. Education of older populations about the identification of early warning signs of melanoma may have the potential to reduce melanoma mortality in this age group.

I05 I06 The Melanoma Epidemic in the U.S. and Australia: Linkage in Time Between Stabilization of Xeroderma Pigmentosum Group C Poly-AT Intron Insertion/Deletion Polymorphism Associated Rates and Decline in Geographic Differences with Prostate Cancer Susceptibility in Normal Donors J. A. H. Lee, R. A. Harrison, D. H. Charache, S.-J. Soong S. G. Khan, V. Bohr, J. Metter, L. Grossman, M. Hedayati, S. Bale, R. Tarone and K. H. Kraemer Department of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, NIH, Bethesda and Baltimore, Maryland and Johns Hopkins School of Public Health (JHSPH), Washington; Biostatistics Unit, Comprehensive Cancer Center, University of Alabama at Baltimore, Maryland Birmingham, Birmingham, Alabama, Consortium for International Earth Science Information Xeroderma pigmentosum patients with defective DNA repair have a 1000-fold increased frequency Network, University Center, Michigan of skin cancers. We are searching for polymorphisms in DNA repair genes that may be present in Incidence and mortality from malignant melanoma in whites are determined by year of birth and the normal population and associated with differences in cancer susceptibility. We sequenced the age (birth-cohort).The differences between successive birth cohorts are diminishing and thus the 3.9kb intron 9 of the XPC gene and found a polymorphism consisting of an insertion of 83 bases incidence and mortality rates are tending to stabilize. This study compares the evolution of the of A and T (poly AT)(PATϩ) plus a 5 base deletion. We developed a rapid PCR based assay to epidemic of fatal melanoma in the US and Australia and investigates the relationship between resolve the PATϩ and PAT– alleles and an actin internal standard using PAGE. We analyzed DNA ultraviolet radiation (UVB) and rates by gender and time period in the U.S. Mortality data for samples from three sources: frozen lymphocytes from JHSPH, lymphocytes from the Baltimore malignant melanoma by gender and time period were abstracted from public use tapes and Longitudinal Study of Aging (BLSA), and buccal swabs from NIH. The frequency of the PATϩ published material. Rates were adjusted to the 1970 World Standard population. Peak instantaneous marker allele was 43.6% in 156 donors with benign skin disorders from JHSPH, 38.6% in 158 LIVI3 values (W per m2) for the population geocentroids of each U.S. state were calculated using cancer-free participants in BLSA, and 35.6% in 216 apparently normal donors from NIH. The a radiative transfer model. The US mortality rates for males and females are increasing rapidly in PATϩ allele frequency increased with age in the JHSPH samples (P ϭ 0.03) but not in the other persons aged 65 and over. Among those aged 45–64 the annual change was 2.7% for males and two groups. The PATϩ allele frequency was 40.9% in 88 donors with basal cell carcinoma from 1.7% for females from 1950 to 1994 in the U.S. and for those aged 15–44 the change was 0.9% JHSPH which was signiﬁcantly higher (P ϭ 0.03) than in 85 skin cancer participants (30.6%) in for males and –0.09% for females. The changes in Australia are similar. For females in the US the the BLSA. The PATϩ frequency was 43.4% in 91 men with prostate cancer in BLSA. However, effect of UVB on melanoma mortality decreased rapidly in all age groups over the period 1968– this population was not in Hardy–Weinberg equilibrium (P ϭ 0.01) and the odds ratio for having 95 (–0.0153). For males, the effect of UVB declined rapidly among those aged 15–44 (–0.0156), prostate cancer and the PATϩ/ϩ genotype was 2.4 [0.9–6.6] compared to 64 age and sex matched and slightly among those aged 65 and over (– 0.001 g). The melanoma epidemics in the US and BLSA controls. The mechanism of the link between PATϩ and prostate cancer susceptibility is Australia are each driven by birth cohort effects that are declining. In spite of their different unknown but may involve alternatively spliced forms of the XPC mRNA. environments, the rates for the two countries are likely to stabilize at levels that are closer than their present ones. The loss of the effect of LIV exposure has contributed substantially to the stabilization of melanoma rates. 664 ABSTRACTS THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

I07 I08 Tanning Facility Usage: are we Exceeding the Limits? Impact of Training in Basic Skin Cancer Triage on Skills and Confidence in Skin Cancer Detection R. L. Hornung, W. J. Lee, L. A. Hansen, A. Patel M. A. Weinstock, M. G. Goldstein, C. E. Dube´ and J. S. Rossi Departments of Dermatology, Pediatrics, Preventive Medicine, and the Institute for Health Services Dermatoepidemiology Unit, VA Medical Center; Department of Dermatology, Rhode Island Research, North-western University Medical School, Chicago, Illinois Hosp; Departments of Dermatology, Psychiatry and Human Behavior, and Community Health, The purpose of this study is to investigate community tanning facility usage, with a particular Brown University; and Cancer Prevention Research Consortium, University of Rhode Island; focus on what extent patrons may be exceeding FDA recommended exposure limits and to Providence and Kingston, Rhode Island quantify how much ultraviolet B and A (UVB/A) they are exposed to during regular usage. Melanoma is a major public health problem for which early detection may reduce mortality. This study involves a community-based survey of tanning facilities and their patrons throughout However, primary care providers (PCPs) receive little training in skin cancer prevention and most the state of North Carolina. Salons were invited to participate as part of their regularly scheduled do not feel confident in their ability to detect skin cancer. We developed a curriculum for PCPs state inspections over a six-month study period. Ten patron records were randomly chosen at to improve their ability to identify potential skin cancers and to triage skin lesions, and sought to each site, and evaluated via a survey instrument consisting of 10 questions pertaining to patron evaluate the effect of a 2-h training with this curriculum on levels of confidence and skills of usage patterns and nine questions pertaining to the tanning facility type and operation. UV PCPs. Provider participation was solicited among the staff of a health maintenance organization; measurements were obtained using a UVB and UVA meter. 28 PCPs participated. Of these, 23 (82%) completed surveys prior to the intervention and one Of the tanning facilities asked to participate, a total of 81% consented to the study. Thus far, the month afterwards. There was a substantial increase in confidence in detection of skin cancer, results indicate that 100% of patrons (N ϭ 243) do not follow the recommended tanning bed assessment of risk, and counseling about primary prevention and early detection (2.9–4.1 on a 1– exposure schedules as outlined by the FDA. Of these, 34% of patrons are actually starting at the 5 scale, P Ͻ 0.0001). Skills were measured by a slide test administered preand post-training, which maximum recommended exposure times. The average time period of tanning for each patron was indicated improvement in diagnostic ability (46% to 64% correct, P Ͻ 0.0001). Substantial though 6.1 wk, and patrons spent approximately 44 min in the tanning bed each week during an average not statistically significant improvements were also noted in triage skills. This brief intervention of 2.4 sessions per week. Seventy-nine percentage of patrons had tanning histories from the for Basic Skin Cancer Triage holds promise for increasing the ability of PCPs to appropriately previous year and were therefore repeat users. As for UV output, the beds on average emitted identify and triage skin cancers. UVB at 5.8 MED per h (range 4.09–9.77 MED per h) and UVA at 16.5 mW per cm2 (range 7.87–25.4 mW per cm2). It is alarmingly clear that FDA recommended exposure schedules are not being followed in the community setting. Current recommendations are based on a gradual increase in exposure time (starting with 0.75 MED/exposure up to a maximum of 4 MED/exposure during the course of several weeks). Furthermore, UV output in standard tanning facility beds is quite high given that, as an example, average UVA output was 165 W per m2 when summer solar noon output is around 50 W per m2. This study indicates a need for future educational interventions to increase the knowledge of both tanning patrons and facility operators in order to comply with recommended exposure guidelines. There may also be a need to further establish enforcement of these safety recommendations.